Stem Cell Derived Factors for Treating Pathologic Conditions

ABSTRACT

A purified paracrine factor of a mesenchymal stem cell, such as a Secreted frizzled related protein (Sfrp) is useful to reduce cell death an/or tissue injury associated with ischemic conditions.

RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.12/726,468, filed Mar. 18, 2010, which is a divisional application ofU.S. Ser. No. 12/008,583, filed Jan. 11, 2008, now U.S. Pat. No.8,129,344, issued Mar. 6, 2012, which is a divisional application ofU.S. Ser. No. 11/508,010, filed Aug. 21, 2006, now U.S. Pat. No.7,638,128, issued Dec. 29, 2009, which claims priority to U.S. Ser. No.60/710,028, filed Aug. 19, 2005, and U.S. Ser. No. 60/711,287, filedAug. 25, 2005, which are incorporated herein by reference in theirentireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. government support under NationalInstitutes of Health grant number HL073219. The government has certainrights in the invention.

FIELD OF THE INVENTION

The invention relates to cardiac disorders.

BACKGROUND OF THE INVENTION

Patient mortality and morbidity is increased by cell/tissue damage ordeath resulting from acute and chronic injury or disease of the heartmuscle, such as myocardial infarction, cardiac failure, stroke,degenerative neurological disease, spinal injury, musculoskeletaldiseases, hypertension, and diabetes.

SUMMARY OF THE INVENTION

The invention is based upon the surprising discovery that paracrinefactors secreted from mesenchymal stem cells (MSC) confer a therapeuticbenefit to bodily tissues. Thus, stem cells serve as a factory ofbiologic products that are purified and administered to subjects.

The paracrine factors are useful in cellular and tissue protection,repair, and regeneration. Mesenchymal stem cells or progenitor cellsthat secrete cytoprotective paracrine factors preferably comprise an Aktgene (Akt-MSC). One or more secreted compounds (e.g., and isolatedcompound or a mixture of secreted compounds such as a MSC culturesupernatant) confers a clinical benefit to a variety of injured,compromised, or disease tissues.

A method of reducing cell death or enhancing tissue repair is carriedout by contacting an injured or diseased tissue with a compositioncomprising a paracrine factor of a mesenchymal stem cell (MSC). Thecomposition is administered to healthy tissue that is determined to beat high risk of injury or to injured tissue following the occurrence ofan injury. Preferably, the factor is a Secreted frizzled related protein(Sfrp). Optionally, the composition contains one or more paracrinefactors, e.g., two, three, five, ten or more factors. The factorsprovide cell reparative benefits in a synergistic manner. For example,the composition contains one or more Sfrp, e.g., Sfrp-1, Sfrp-2, andSfrp-3. In one embodiment, Sfrp-1 comprises an amino acid sequence ofSEQ ID NO:5, a mature processed form of SEQ ID NO:5, or a fragmentthereof; in another embodiment, Sfrp-2 comprises an amino acid sequenceof SEQ ID NO:7, a mature processed form of SEQ ID NO:7, or a fragmentthereof; and in yet another embodiment, Sfrp-3 comprises an amino acidsequence of SEQ ID NO:9, a mature processed form of SEQ ID NO:9, or afragment thereof. The amount of apoptotic cell death is reduced in thepresence of a paracrine factor such as an Sfrp compared to in itsabsence.

Cytoprotective and cell reparative effects are conferred to many typesof bodily tissues such as cardiac tissue. For example, in the case of amyocardial infarction, cardiac infarct size is reduced following contactof myocardial tissue with the paracrine factor.

Factors derived from Akt-MSCs, which have been genetically altered tocontain a recombinant Akt gene sequence, confer a therapeutic benefit ateach stage of a hypoxic cardiac event (early, middle, and late stage).Early on, factors confer a cell protective effect, followed by inotropy,angiogenesis, and cardiac remodeling.

The invention also features methods of inhibiting cell damage, inducingor enhancing cell repair or regeneration or inhibiting an ischemic orreperfusion related injury in a subject. Cell damage or injury isinhibited by administering to the subject or contacting a cell with acomposition containing a purified cytoprotective compound such as asubstantially pure polypeptide, or a mixture of substantially purepolypeptides such as the Sfrp proteins described above. Other purifiedproteins, e.g., h1, h5, h8, h12, and h13 are also useful to prevent orreduce cell damage. Accordingly, a method of reducing cell death iscarried out by contacting an injured or diseased tissue with acomposition comprising a purified paracrine factor of a mesenchymal stemcell selected from the group consisting of h1, h5, h8, h12 and h13 orfragment thereof. For example, h12 comprises a fragment of SEQ ID NO:17.

Similarly, cell repair or regeneration is induced by administering tothe subject or contacting a cell with a composition containing apurified cytoprotective compound. Polypeptides or other compoundsdescribed herein are said to be “substantially pure” when they arewithin preparations that are at least 60% by weight (dry weight) thecompound of interest. Preferably, the preparation is at least 75%, morepreferably at least 90%, and most preferably at least 99%, by weight thecompound of interest. Purity is measured by any appropriate standardmethod, for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis. The polypeptide is purified from MSCculture media or recombinantly produced.

Cell or tissue damage is defined by a loss or diminution of cellfunction. Such loss or decrease in function leads to eventual celldeath. The cell is a cardiac cell such as a cardiomyocyte, a kidneycell, a liver cell, a neurological (e.g., brain, spinal cord) cell, or apancreatic cell. For example, a loss of cardiomyocyte function resultsin the loss of the contractile function of the cell. Cardiomyocytes thathave lost their ability to contract form round cells rather that rodshaped cells when cultured. Ischemia causes irreversible cellular/tissuedamage and cell death. Reperfusion exacerbates ischemic damage byactivating inflammatory response and oxidative stress. Oxidative stressmodifies membrane lipids, proteins and nucleic acids resulting incellular/tissue damage or death, and depression of cardiac, endothelialand kidney function.

Also included in the invention are methods of regenerating an injuredmyocardial tissue by administered to the tissue a composition containinga cytoprotective compound. The cardiac muscle has been damaged bydisease, such as a myocardial infarction. By regenerating an injuredmyocardial tissue is meant restoring ventricular function and/ordecreasing infarct size. Ventricular function is measured by methodsknown in the art such as radionuclide angiography.

A cytoprotective compound is a compound, which is capable of inhibitingcell damage such as oxidative-stress induced cell death or apoptosis. Inaddition to Sfrps, cytoprotective compounds include for example adipsin,adrenomedullin, chemokine (C—C motif) ligand 2, cysteine rich protein61, lysyl oxidase-like 2, or serine proteinase inhibitor.

The composition is administered to the subject prior to, at the time of,or shortly after (1, 5, 10, 15, 30, 60 minutes; 1.5, 2, 4, 6, 12, 18,24, 48 hours) identification of cell damage or identification of asymptom of ischemia or reperfusion injury. For example the compositionis administered to a subject prior to a cardiac event orischemic-reperfusion injury. Such a subject is a risk candidate for anischemic event or condition. Symptoms of a cardiac event include forexample, chest pain, arm pain, fatigue and shortness of breath. Forexample, the composition is administered at the onset of symptoms, e.g.,chest pain, associated with a cardiac event such as a myocardialinfarction. The composition is administered systemically or locally. Forexample, the composition is administered directly, i.e., by myocardialinjection to the cardiac tissue, or systemically, e.g.,interperitoneally, orally, intravenously. In another example,administration of the composition is carried out by infusion into acoronary artery. Slow-release formulations, e.g., a dermal patch, inwhich diffusion of the composition from an excipient such as a polymericcarrier mediates drug delivery are also within the invention.Optionally, the subject is further administered VEGF or thyrosin beta 4.

The composition is administered at a dose sufficient to inhibitapoptotic death or oxidative stress-induced cell death. To determinewhether the composition inhibits oxidative-stress induced cell death,the composition is tested by incubating the composition with a primaryor immortalized cell such as a cardiomyocyte. A state of oxidativestress of the cells is induced (e.g., by incubating cells with H₂O₂),and cell viability is measured using standard methods. As a control, thecells are incubated in the absence of the composition and then a stateof oxidative stress is induced. A decrease in cell death (or an increasein the number of viable cells) in the compound treated sample indicatesthat the composition inhibits oxidative-stress induced cell death.Alternatively, an increase in cell death (or an decrease in the numberof viable cells) in the compound treated sample indicates that thecomposition does not inhibit oxidative-stress induced cell death. Thetest is repeated using different doses of the composition to determinethe dose range in which the composition functions to inhibitoxidative-stress induced cell death.

A subject to be treated is suffering from or at risk of developing acondition characterized by aberrant cell damage such as oxidative-stressinduced cell death (e.g., apoptotic cell death) or an ischemic orreperfusion related injury. A subject suffering from or at risk ofdeveloping such a condition is identified by the detection of a knownrisk factor, e.g., gender, age, high blood pressure, obesity, diabetes,prior history of smoking, stress, genetic or familial predisposition,attributed to the particular disorder, or previous cardiac event such asmyocardial infarction or stroke.

Conditions characterized by aberrant cell damage or death includecardiac disorders (acute or chronic) such as stroke, myocardialinfarction, chronic coronary ischemia, arteriosclerosis, congestiveheart failure, dilated cardiomyopathy, restenosis, coronary arterydisease, heart failure, arrhythmia, angina, atherosclerosis,hypertension, renal failure, kidney ischemia, ischemic hepatitis,hepatic vein thrombosis, cirrhosis, portal vein thrombosis,pancreatitis, ischemic colitis, or myocardial hypertrophy. Cardiacrepair or regeneration is evaluated by detecting an improvement ofsymptoms such as chest pain or shortness of breath as well as byevaluation of heart function by standard methods such as cardiacmagnetic resonance, echocardiography, and/or ventricular angiography.

Also within the invention is a cell culture or preservation mediacontaining purified Sfrp2 and a method of maintaining inhibiting stemcell differentiation, e.g., inhibiting myogenesis, by contacting apopulation of isolated stem cells with purified Sfrp2. Isolated stemcells are selected from the group consisting of embryonic stem cells,mesenchymal stem cells, and hematopoetic stem cells. Stem cells areisolated from the tissue of origin by fractionation by cell surfacemarkers or other distinguishing characteristics. Preferably, apopulation of isolated cells is at least 85% stem cells. Morepreferably, the population is 90, 95, 98, 99, 100% stem cells.

This factor is involved in the maintenance and self renewal of tissuespecific and embryonic stem cells. For example, differentiation of stemcells, e.g., embryonic stem cells, is inhibited by Sfrp2. Myogenesis isinhibited by contacting stem cells with Sfrp2. In another example, bonemarrow-derived hematopoetic stem are maintained in a stem cell state bycontacting the cells with purified Sfrp2. Preservation of stem cells inthis manner is useful in transport and storage of stem cells prior totransplantation into a subject for therapeutic purposes.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are bar graphs showing that Sfrps are expressed inmesenchymal stem cells. FIG. 1A shows levels of Sfrp1, Sfrp2 and Sfrp3expression as estimated by microarray analysis and shows a nearly 10fold upregulation of Sfrp2 in Akt-MSC compared to GFP-MSC. FIG. 1B showsa quantitative real-time RT-PCR validation of mRNA to expression levelsthat demonstrates a 100 fold upregulation of Sfrp2 gene expression inAkt-MSC compared to GFP-MSC.

FIG. 2A is a photograph of results of a Western Blotting assay forSfrp2. The data demonstrates presence of Sfrp2 protein in conditionedmedium collected from AKT or GFP MSCs and inhibition of its accumulationin the medium in the presence of Pi3K inhibitor

FIG. 2B is a bar graph showing relative reduction in mRNA levels ofSfrp2 in Akt-MSC following knockdown of Sfrp2 with siRNA.

FIG. 2C is a bar graph showing the effect of conditioned medium onapoptosis in ARVCs. Caspase activity of ARVCs after 24 hours of hypoxiaunder different culture conditions (control conditioned medium, Ctr CM;Akt conditioned medium, Akt CM; Akt conditioned medium following Sfrp2knockdown, Akt CM minus Sfrp2) demonstrates reduction of caspaseactivity following Akt-CM treatment and attenuation of this effectfollowing treatment with Akt CM minus Sfrp2. These data demonstrate thatparacrine factors from Akt-MSCs mediate the survival signaling oncardiomycytes.

FIG. 3A is a bar graph showing the effect of Sfrp2 on caspase activity.Cleaved-caspase 3 activity as measured by a fluorometric assaydemonstrated decreased caspase activity in hypoxic cardiomyocytesfollowing Sfrp2 treatment in a dose dependent manner. The activity wascalculated as fold changes with the same control.

FIG. 3B is a bar graph showing the number of round shaped cardiomyocytesthat were counted in 6 random high power fields (40×) following 24 hourhypoxic exposure with/without Sfrp2 treatment. Data is expressed as apercentage of total number of cells present.

FIG. 3C is a series of representative high power field photographsdemonstrating decreased number of round shaped cardiomyocytes followingtreatment with Sfrp2. Collectively, these data demonstrate that Sfrp2decreases caspase 3 activity

FIG. 4 is a bar graph showing that Sfrp2 decreased cardiac infarct size.Above each bar, is a photograph of TTC staining showing bi-ventricularsections of similar thickness perpendicular to the long axis of theheart. The staining data demonstrates decreased infarct size with Akt-CMand Sfrp2 and attenuation of reduction in infarct size with Akt-Sfrp2.Infarct size is expressed as a percentage of the total ventricular area.Rat hearts were treated with PBS as control, Akt-MSCs CM (Akt), CM formAkt-MSCs that did express reduced to levels of sfrp2 due to siRNAtreatment (Akt-Sfrp2).

FIG. 5A is a photograph of an electrophoretic gel showing that Wnt3amRNA expression as detected by RT-PCR is increased in hypoxiccardiomyocytes while expression of Wnt5 remains unchanged. The dataindicate that hypoxic cardiomyocytes upregulate Wnt3a expression andthat Sfrp2 blocks pro-apototic effects of Wnt3a.

FIG. 5B is a bar graph showing that Wnt3a (3 nM) increases caspaseactivity of cardiomyocytes undergoing hypoxia/reoxygenation injury;Sfrp2 at a similar concentration significantly attenuates Wnt3a inducedcaspase activity (* vs. normoxia, p<0.05; ** vs.wnt+hypoxia/reoxygenation, p<0.05, n=6/group).

FIG. 6A is a bar graph showing genes upregulated by Sfrp2 under hypoxia.Microarray analysis demonstrates Sfrp2 mediated upregulation of Birc1bgene expression in hypoxic cardiomyocytes.

FIG. 6B is a photograph of an electrophoretic gel showing the effect ofSfrp2 on mRNA levels on Birc1b. RT-PCR confirmed increased Birc1bexpression in hypoxic cardiomyocytes following Sfrp2 treatment.

FIG. 6C is a photograph of results of a Western Blot showing thatbeta-catenin levels are increased by Sfrp2. Western blotting for nuclearand total βcatenin expression in ARVCs demonstrates a reduction ofβcatenin following hypoxia and upregulation following treatment withSfrp2.

FIG. 7 is a bar graph showing the effect of cytoprotective factor h12compared to IGF-1 on myocyte apoptosis.

FIG. 8 is a line graph showing caspase inhibition in cardiomyocytes byh12.

FIG. 9 is a series of photographs electrophoretic gels showing that h12phosphorylates/activates AKT in cardiomyocytes

FIG. 10 is a photograph showing inhibition of cytochrome C release byh12.

FIG. 11 is a photograph of an electrophoretic gel showing mitochondrialBcl-2 protein stabilization by h12.

DETAILED DESCRIPTION

The present invention is based upon the unexpected discovery of thatMSC-secreted products confer a therapeutic benefit to injured orcompromised tissues. Disclosed herein is a Akt-MSC mediated paracrinemechanism of organ protection and repair. More particularly, theinvention provides purified polypeptides such as Srfps isolated fromAkt-MSCs or recombinantly or synthetically produced and methods of usingthese polypeptides to prevent or reduce myocardial damage andventricular dysfunction.

Akt Genes

Akt-MSCs are produced by introducing (e.g., by retrovirus-mediatedtransduction) into mesenchymal stem cells isolated from the bone marrowan Akt coding sequence or fragment, e.g., Akt-1, Ak-2 or Akt-3. The Aktnucleic acid is human, mouse, or rat.

Exemplary human Akt-1 polypeptides include GenBank Accession numbersNP_(—)005154 and AAH00479. Exemplary human Akt-2 polypeptides includesfor example GenBank Accession numbers P31751 and NP_(—)001617. Exemplaryhuman Akt-3 polypeptides includes for example GenBank Accession numbersQ9Y243 and NP_(—)005456. Exemplary nucleic acids encoding Akt includehuman Akt-1 available at GENBANK™ Accession No. NM 005163 (SEQ ID NO:1),human Akt-2 available at GENBANK™ Accession No. NM 001626 (SEQ ID NO:2)and human Akt-3 available at GENBANK™ Accession No. AJ245709 (SEQ IDNO:3) (all of which are hereby incorporated by reference) or nucleicacids encoding the human Akt polypeptides described above. mRNAsequences and the corresponding coding region for human Akt are shownbelow.

Akt-1 mRNA (SEQ ID NO: 1) 1atcctgggac agggcacagg gccatctgtc accaggggct tagggaaggc cgagccagcc 61tgggtcaaag aagtcaaagg ggctgcctgg aggaggcagc ctgtcagctg gtgcatcaga 121ggctgtggcc aggccagctg ggctcgggga gcgccagcct gagaggagcg cgtgagcgtc 181gcgggagcct cgggcaccat gagcgacgtg gctattgtga aggagggttg gctgcacaaa 241cgaggggagt acatcaagac ctggcggcca cgctacttcc tcctcaagaa tgatggcacc 301ttcattggct acaaggagcg gccgcaggat gtggaccaac gtgaggctcc cctcaacaac 361ttctctgtgg cgcagtgcca gctgatgaag acggagcggc cccggcccaa caccttcatc 421atccgctgcc tgcagtggac cactgtcatc gaacgcacct tccatgtgga gactcctgag 481gagcgggagg agtggacaac cgccatccag actgtggctg acggcctcaa gaagcaggag 541gaggaggaga tggacttccg gtcgggctca cccagtgaca actcaggggc tgaagagatg 601gaggtgtccc tggccaagcc caagcaccgc gtgaccatga acgagtttga gtacctgaag 661ctgctgggca agggcacttt cggcaaggtg atcctggtga aggagaaggc cacaggccgc 721tactacgcca tgaagatcct caagaaggaa gtcatcgtgg ccaaggacga ggtggcccac 781acactcaccg agaaccgcgt cctgcagaac tccaggcacc ccttcctcac agccctgaag 841tactctttcc agacccacga ccgcctctgc tttgtcatgg agtacgccaa cgggggcgag 901ctgttcttcc acctgtcccg ggaacgtgtg ttctccgagg accgggcccg cttctatggc 961gctgagattg tgtcagccct ggactacctg cactcggaga agaacgtggt gtaccgggac 1021ctcaagctgg agaacctcat gctggacaag gacgggcaca ttaagatcac agacttcggg 1081ctgtgcaagg aggggatcaa ggacggtgcc accatgaaga ccttttgcgg cacacctgag 1141tacctggccc ccgaggtgct ggaggacaat gactacggcc gtgcagtgga ctggtggggg 1201ctgggcgtgg tcatgtacga gatgatgtgc ggtcgcctgc ccttctacaa ccaggaccat 1261gagaagcttt ttgagctcat cctcatggag gagatccgct tcccgcgcac gcttggtccc 1321gaggccaagt ccttgctttc agggctgctc aagaaggacc ccaagcagag gcttggcggg 1381ggctccgagg acgccaagga gatcatgcag catcgcttct ttgccggtat cgtgtggcag 1441cacgtgtacg agaagaagct cagcccaccc ttcaagcccc aggtcacgtc ggagactgac 1501accaggtatt ttgatgagga gttcacggcc cagatgatca ccatcacacc acctgaccaa 1561gatgacagca tggagtgtgt ggacagcgag cgcaggcccc acttccccca gttctcctac 1621tcggccagca gcacggcctg aggcggcggt ggactgcgct ggacgatagc ttggagggat 1681ggagaggcgg cctcgtgcca tgatctgtat ttaatggttt ttatttctcg ggtgcatttg 1741agagaagcca cgctgtcctc tcgagcccag atggaaagac gtttttgtgc tgtgggcagc 1801accctccccc gcagcggggt agggaagaaa actatcctgc gggttttaat ttatttcatc 1861cagtttgttc tccgggtgtg gcctcagccc tcagaacaat ccgattcacg tagggaaatg 1921ttaaggactt ctacagctat gcgcaatgtg gcattggggg gccgggcagg tcctgcccat 1981gtgtcccctc actctgtcag ccagccgccc tgggctgtct gtcaccagct atctgtcatc 2041tctctggggc cctgggcctc agttcaacct ggtggcacca gatgcaacct cactatggta 2101tgctggccag caccctctcc tgggggtggc aggcacacag cagcccccca gcactaaggc 2161cgtgtctctg aggacgtcat cggaggctgg gcccctggga tgggaccagg gatgggggat 2221gggccagggt ttacccagtg ggacagagga gcaaggttta aatttgttat tgtgtattat 2281gttgttcaaa tgcattttgg gggtttttaa tctttgtgac aggaaagccc tcccccttcc 2341ccttctgtgt cacagttctt ggtgactgtc ccaccggagc ctccccctca gatgatctct 2401ccacggtagc acttgacctt ttcgacgctt aacctttccg ctgtcgcccc aggccctccc 2461tgactccctg tgggggtggc catccctggg cccctccacg cctcctggcc agacgctgcc 2521gctgccgctg caccacggcg tttttttaca acattcaact ttagtatttt tactattata 2581atataatatg gaaccttccc tccaaattct Coding sequence = nucleotide 199-1641.Akt-2 mRNA (SEQ ID NO: 2) 1gaattccagc ggcggcgccg ttgccgctgc cgggaaacac aaggaaaggg aaccagcgca 61gcgtggcgat gggcgggggt agagccccgc cggagaggct gggcggctgc cggtgacaga 121ctgtgccctg tccacggtgc ctcctgcatg tcctgctgcc ctgagctgtc ccgagctagg 181tgacagcgta ccacgctgcc accatgaatg aggtgtctgt catcaaagaa ggctggctcc 241acaagcgtgg tgaatacatc aagacctgga ggccacggta cttcctgctg aagagcgacg 301gctccttcat tgggtacaag gagaggcccg aggcccctga tcagactcta ccccccttaa 361acaacttctc cgtagcagaa tgccagctga tgaagaccga gaggccgcga cccaacacct 421ttgtcatacg ctgcctgcag tggaccacag tcatcgagag gaccttccac gtggattctc 481cagacgagag ggaggagtgg atgcgggcca tccagatggt cgccaacagc ctcaagcagc 541gggccccagg cgaggacccc atggactaca agtgtggctc ccccagtgac tcctccacga 601ctgaggagat ggaagtggcg gtcagcaagg cacgggctaa agtgaccatg aatgacttcg 661actatctcaa actccttggc aagggaacct ttggcaaagt catcctggtg cgggagaagg 721ccactggccg ctactacgcc atgaagatcc tgcgaaagga agtcatcatt gccaaggatg 781aagtcgctca cacagtcacc gagagccggg tcctccagaa caccaggcac ccgttcctca 841ctgcgctgaa gtatgccttc cagacccacg accgcctgtg ctttgtgatg gagtatgcca 901acgggggtga gctgttcttc cacctgtccc gggagcgtgt cttcacagag gagcgggccc 961ggttttatgg tgcagagatt gtctcggctc ttgagtactt gcactcgcgg gacgtggtat 1021accgcgacat caagctggaa aacctcatgc tggacaaaga tggccacatc aagatcactg 1081actttggcct ctgcaaagag ggcatcagtg acggggccac catgaaaacc ttctgtggga 1141ccccggagta cctggcgcct gaggtgctgg aggacaatga ctatggccgg gccgtggact 1201ggtgggggct gggtgtggtc atgtacgaga tgatgtgcgg ccgcctgccc ttctacaacc 1261aggaccacga gcgcctcttc gagctcatcc tcatggaaga gatccgcttc ccgcgcacgc 1321tcagccccga ggccaagtcc ctgcttgctg ggctgcttaa gaaggacccc aagcagaggc 1381ttggtggggg gcccagcgat gccaaggagg tcatggagca caggttcttc ctcagcatca 1441actggcagga cgtggtccag aagaagctcc tgccaccctt caaacctcag gtcacgtccg 1501aggtcgacac aaggtacttc gatgatgaat ttaccgccca gtccatcaca atcacacccc 1561ctgaccgcta tgacagcctg ggcttactgg agctggacca gcggacccac ttcccccagt 1621tctcctactc ggccagcatc cgcgagtgag cagtctgccc acgcagagga cgcacgctcg 1681ctgccatcac cgctgggtgg ttttttaccc ctgcc Coding sequence = nucleotide204-1649. Akt-3 mRNA (SEQ ID NO:3) 1gggagtcatc atgagcgatg ttaccattgt gaaagaaggt tgggttcaga agaggggaga 61atatataaaa aactggaggc caagatactt ccttttgaag acagatggct cattcatagg 121atataaagag aaacctcaag atgtggattt accttatccc ctcaacaact tttcagtggc 181aaaatgccag ttaatgaaaa cagaacgacc aaagccaaac acatttataa tcagatgtct 241ccagtggact actgttatag agagaacatt tcatgtagat actccagagg aaagggaaga 301atggacagaa gctatccagg ctgtagcaga cagactgcag aggcaagaag aggagagaat 361gaattgtagt ccaacttcac aaattgataa tataggagag gaagagatgg atgcctctac 421aacccatcat aaaagaaaga caatgaatga ttttgactat ttgaaactac taggtaaagg 481cacttttggg aaagttattt tggttcgaga gaaggcaagt ggaaaatact atgctatgaa 541gattctgaag aaagaagtca ttattgcaaa ggatgaagtg gcacacactc taactgaaag 601cagagtatta aagaacacta gacatccctt tttaacatcc ttgaaatatt ccttccagac 661aaaagaccgt ttgtgttttg tgatggaata tgttaatggg ggcgagctgt ttttccattt 721gtcgagagag cgggtgttct ctgaggaccg cacacgtttc tatggtgcag aaattgtctc 781tgccttggac tatctacatt ccggaaagat tgtgtaccgt gatctcaagt tggagaatct 841aatgctggac aaagatggcc acataaaaat tacagatttt ggactttgca aagaagggat 901cacagatgca gccaccatga agacattctg tggcactcca gaatatctgg caccagaggt 961gttagaagat aatgactatg gccgagcagt agactggtgg ggcctagggg ttgtcatgta 1021tgaaatgatg tgtgggaggt tacctttcta caaccaggac catgagaaac tttttgaatt 1081aatattaatg gaagacatta aatttcctcg aacactctct tcagatgcaa aatcattgct 1141ttcagggctc ttgataaagg atccaaataa acgccttggt ggaggaccag atgatgcaaa 1201agaaattatg agacacagtt tcttctctgg agtaaactgg caagatgtat atgataaaaa 1261gcttgtacct ccttttaaac ctcaagtaac atctgagaca gatactagat attttgatga 1321agaatttaca gctcagacta ttacaataac accacctgaa aaatatgatg aggatggtat 1381ggactgcatg gacaatgaga ggcggccgca tttccctcaa ttttcctact ctgcaagtgg 1441acgagaataa gtctctttca ttctgctact tcactgtcat cttcaattta ttactgaaaa 1501tgattcctgg acatcaccag tcctagctct tacacatagc aggggca Coding sequence =nucleotide 11-1450

Intramyocardial transplantation of adult stem cells has been proposed asa therapy to repair and regenerate damaged myocardium and to restorecardiac function after acute myocardial infarction (MI). Given theirmultipotency, low immunogenicity, amenability to ex vivo expansion andgenetic modification, autologous bone marrow derived MSCs are suitablefor this purpose. Injection of MSCs reduces post-infarction ventricularremodeling and in some cases improves left ventricular function. Howeverprior to the invention, mechanism(s) underlying these therapeuticeffects have not been clearly defined. In situ differentiation of thetransplanted MSCs into cardiomyocytes and other constituent cardiac celltypes has been suggested. Intramyocardial transplantation of MSCstransduced with a retroviral vector overexpressing the survival gene Aktmarkedly improves the therapeutic efficacy of MSCs in preventingventricular remodeling and restoring cardiac function.

The data described herein shows that therapeutic effects seen with theadministration of cells occur in less than 72 hours after infarction.These early dramatic effects cannot be readily attributed to myocardialregeneration or neoangiogenesis, but rather indicate that Akt-MSCsrelease biologically active factors that exert paracrine actions on theischemic cardiomyocytes. Under hypoxic stimulation, genetically-modifiedbone marrow derived MSCs overexpressing the Akt gene release paracrinefactors that exert cytoprotective effects on isolated cardiomyocytes.Intramyocardial injection of these substances reduces infarct size,prevents left ventricular dysfunction, and decreases in the number ofapoptotic cardiomyocytes in vivo. In addition, no increase inmicrovessel density was observed in is the treated groups compared tocontrols 72 hours after the injection of the conditioned medium Thus, asignificant portion of the salutary effects of Akt-MSCs transplantationis attributable to protection and functional recovery of ischemicmyocardium, instead of, or in addition to, de novo cardiac repair andregeneration. The ability of bone marrow derived MSCs, especiallyAkt-MSCs, to produce factor(s) capable of protecting cardiomyocytes fromcell death has not been previously demonstrated.

Secreted Frizzled-Related Proteins

The GENBANK™ Accession numbers of human Sfrps include BCO36503 (Sfrp1),BC008666 (Sfrp2), and NM001463 (Sfrp3), hereby incorporated byreference. The amino acid sequence of exemplary Sfrp polypeptides andnucleotides encoding the polypeptides (coding sequences) are describedbelow. The Sfrp polypeptides, mature processed forms, and/or fragmentsthereof are used in the cardioprotective and repair methods describedherein.

Human SFRP1 mRNA sequence (SEQ ID NO: 4) 1cctgcagcct ccggagtcag tgccgcgcgc ccgccgcccc gcgccttcct gctcgccgca 61cctccgggag ccggggcgca cccagcccgc agcgccgcct ccccgcccgc gccgcctccg 121accgcaggcc gagggccgcc actggccggg gggaccgggc agcagcttgc ggccgcggag 181ccgggcaacg ctggggactg cgccttttgt ccccggaggt ccctggaagt ttgcggcagg 241acgcgcgcgg ggaggcggcg gaggcagccc cgacgtcgcg gagaacaggg cgcagagccg 301gcatgggcat cgggcgcagc gaggggggcc gccgcggggc agccctgggc gtgctgctgg 361cgctgggcgc ggcgcttctg gccgtgggct cggccagcga gtacgactac gtgagcttcc 421agtcggacat cggcccgtac cagagcgggc gcttctacac caagccacct cagtgcgtgg 481acatccccgc ggacctgcgg ctgtgccaca acgtgggcta caagaagatg gtgctgccca 541acctgctgga gcacgagacc atggcggagg tgaagcagca ggccagcagc tgggtgcccc 601tgctcaacaa gaactgccac gccggcaccc aggtcttcct ctgctcgctc ttcgcgcccg 661tctgcctgga ccggcccatc tacccgtgtc gctggctctg cgaggccgtg cgcgactcgt 721gcgagccggt catgcagttc ttcggcttct actggcccga gatgcttaag tgtgacaagt 781tccccgaggg ggacgtctgc atcgccatga cgccgcccaa tgccaccgaa gcctccaagc 841cccaaggcac aacggtgtgt cctccctgtg acaacgagtt gaaatctgag gccatcattg 901aacatctctg tgccagcgag tttgcactga ggatgaaaat aaaagaagtg aaaaaagaaa 961atggcgacaa gaagattgtc cccaagaaga agaagcccct gaagttgggg cccatcaaga 1021agaaggacct gaagaagctt gtgctgtacc tgaagaatgg ggctgactgt ccctgccacc 1081agctggacaa cctcagccac cacttcctca tcatgggccg caaggtgaag agccagtact 1141tgctgacggc catccacaag tgggacaaga aaaacaagga gttcaaaaac ttcatgaaga 1201aaatgaaaaa ccatgagtgc cccacctttc agtccgtgtt taagtgattc tcccgggggc 1261agggtgggga gggagcctcg ggtggggtgg gagcgggggg gacagtgccc cgggaacccg 1321gtgggtcaca cacacgcact gcgcctgtca gtagtggaca ttgtaatcca gtcggcttgt 1381tcttgcagca ttcccgctcc cttccctcca tagccacgct ccaaacccca gggtagccat 1441ggccgggtaa agcaagggcc atttagatta ggaaggtttt taagatccgc aatgtggagc 1501agcagccact gcacaggagg aggtgacaaa ccatttccaa cagcaacaca gccactaaaa 1561cacaaaaagg gggattgggc ggaaagtgag agccagcagc aaaaactaca ttttgcaact 1621tgttggtgtg gatctattgg ctgatctatg cctttcaact agaaaattct aatgattggc 1681aagtcacgtt gttttcaggt ccagagtagt ttctttctgt ctgctttaaa tggaaacaga 1741ctcataccac acttacaatt aaggtcaagc ccagaaagtg ataagtgcag ggaggaaaag 1801tgcaagtcca ttatgtaata gtgacagcaa agggaccagg ggagaggcat tgccttctct 1861gcccacagtc tttccgtgtg attgtctttg aatctgaatc agccagtctc agatgcccca 1921aagtttcggt tcctatgagc ccggggcatg atctgatccc caagacatgt ggaggggcag 1981cctgtgcctg cctttgtgtc agaaaaagga aaccacagtg agcctgagag agacggcgat 2041tttcgggctg agaaggcagt agttttcaaa acacatagtt aaaaaagaaa caaatgaaaa 2101aaattttaga acagtccagc aaattgctag tcagggtgaa ttgtgaaatt gggtgaagag 2161cttaggattc taatctcatg ttttttcctt ttcacatttt taaaagaaca atgacaaaca 2221cccacttatt tttcaaggtt ttaaaacagt ctacattgag catttgaaag gtgtgctaga 2281acaaggtctc ctgatccgtc cgaggctgct tcccagagga gcagctctcc ccaggcattt 2341gccaagggag gcggatttcc ctggtagtgt agctgtgtgg ctttccttcc tgaagagtcc 2401gtggttgccc tagaacctaa caccccctag caaaactcac agagctttcc gtttttttct 2461ttcctgtaaa gaaacatttc ctttgaactt gattgcctat ggatcaaaga aattcagaac 2521agcctgcctg tccccccgca ctttttacat atatttgttt catttctgca gatggaaagt 2581tgacatgggt ggggtgtccc catccagcga gagagtttca aaagcaaaac atctctgcag 2641tttttcccaa gtaccctgag atacttccca aagcccttat gtttaatcag cgatgtatat 2701aagccagttc acttagacaa ctttaccctt cttgtccaat gtacaggaag tagttctaaa 2761aaaaatgcat attaatttct tcccccaaag ccggattctt aattctctgc aacactttga 2821ggacatttat gattgtccct ctgggccaat gcttataccc agtgaggatg ctgcagtgag 2881gctgtaaagt ggccccctgc ggccctagcc tgacccggag gaaaggatgg tagattctgt 2941taactcttga agactccagt atgaaaatca gcatgcccgc ctagttacct accggagagt 3001tatcctgata aattaacctc tcacagttag tgatcctgtc cttttaacac cttttttgtg 3061gggttctctc tgacctttca tcgtaaagtg ctggggacct taagtgattt gcctgtaatt 3121ttggatgatt aaaaaatgtg tatatatatt agctaattag aaatattcta cttctctgtt 3181gtcaaactga aattcagagc aagttcctga gtgcgtggat ctgggtctta gttctggttg 3241attcactcaa gagttcagtg ctcatacgta tctgctcatt ttgacaaagt gcctcatgca 3301accgggccct ctctctgcgg cagagtcctt agtggagggg tttacctgga acattagtag 3361ttaccacaga atacggaaga gcaggtgact gtgctgtgca gctctctaaa tgggaattct 3421caggtaggaa gcaacagctt cagaaagagc tcaaaataaa ttggaaatgt gaatcgcagc 3481tgtgggtttt accaccgtct gtctcagagt cccaggacct tgagtgtcat tagttacttt 3541attgaaggtt ttagacccat agcagctttg tctctgtcac atcagcaatt tcagaaccaa 3601aagggaggct ctctgtaggc acagagctgc actatcacga gcctttgttt ttctccacaa 3661agtatctaac aaaaccaatg tgcagactga ttggcctggt cattggtctc cgagagagga 3721ggtttgcctg tgatttccta attatcgcta gggccaaggt gggatttgta aagctttaca 3781ataatcattc tggatagagt cctgggaggt ccttggcaga actcagttaa atctttgaag 3841aatatttgta gttatcttag aagatagcat gggaggtgag gattccaaaa acattttatt 3901tttaaaatat cctgtgtaac acttggctct tggtacctgt gggttagcat caagttctcc 3961ccagggtaga attcaatcag agctccagtt tgcatttgga tgtgtaaatt acagtaatcc 4021catttcccaa acctaaaatc tgtttttctc atcagactct gagtaactgg ttgctgtgtc 4081ataacttcat agatgcagga ggctcaggtg atctgtttga ggagagcacc ctaggcagcc 4141tgcagggaat aacatactgg ccgttctgac ctgttgccag cagatacaca ggacatggat 4201gaaattcccg tttcctctag tttcttcctg tagtactcct cttttagatc ctaagtctct 4261tacaaaagct ttgaatactg tgaaaatgtt ttacattcca tttcatttgt gttgtttttt 4321taactgcatt ttaccagatg ttttgatgtt atcgcttatg ttaatagtaa ttcccgtacg 4381tgttcatttt attttcatgc tttttcagcc atgtatcaat attcacttga ctaaaatcac 4441tcaattaatc aatgaaaaaa aaaaa Human SFRP1 protein sequence (SEQ ID NO: 5)MGIGRSEGGRRGAALGVLLALGAALLAVGSASEYDYVSFQSDIGPYQSGRFYTKPPQCVDIPADLRLCHNVGYKKMVLPNLLEHETMAEVKQQASSWVPL LNKNCHAGTQVFLCSLFAPVCLDRPIYPCRWLCEAVRDSCEPVMQFFGFYWPEMLKCD KFPEGDVCIAMTPPNATEASKPQGTTVCPPCDNELKSEAIIEHLCASEFALRMKIKEVKKENGDKKIVPKKKKPLKLGPIKKKDLKKLVLYLKNGADCPCHQLDNLSHHFLIMGRKVK        SQYLLTAIHKWDKKNKEFKNFMKKMKNHECPTFQSVFKHuman SFRP2 mRNA sequence (SEQ ID NO: 6) 1caacggctca ttctgctccc ccgggtcgga gccccccgga gctgcgcgcg ggcttgcagc 61gcctcgcccg cgctgtcctc ccggtgtccc gcttctccgc gccccagccg ccggctgcca 121gcttttcggg gccccgagtc gcacccagcg aagagagcgg gcccgggaca agctcgaact 181ccggccgcct cgcccttccc cggctccgct ccctctgccc cctcggggtc gcgcgcccac 241gatgctgcag ggccctggct cgctgctgct gctcttcctc gcctcgcact gctgcctggg 301ctcggcgcgc gggctcttcc tctttggcca gcccgacttc tcctacaagc gcagcaattg 361caagcccatc cctgccaacc tgcagctgtg ccacggcatc gaataccaga acatgcggct 421gcccaacctg ctgggccacg agaccatgaa ggaggtgctg gagcaggccg gcgcttggat 481cccgctggtc atgaagcagt gccacccgga caccaagaag ttcctgtgct cgctcttcgc 541ccccgtctgc ctcgatgacc tagacgagac catccagcca tgccactcgc tctgcgtgca 601ggtgaaggac cgctgcgccc cggtcatgtc cgccttcggc ttcccctggc ccgacatgct 661tgagtgcgac cgtttccccc aggacaacga cctttgcatc cccctcgcta gcagcgacca 721cctcctgcca gccaccgagg aagctccaaa ggtatgtgaa gcctgcaaaa ataaaaatga 781tgatgacaac gacataatgg aaacgctttg taaaaatgat tttgcactga aaataaaagt 841gaaggagata acctacatca accgagatac caaaatcatc ctggagacca agagcaagac 901catttacaag ctgaacggtg tgtccgaaag ggacctgaag aaatcggtgc tgtggctcaa 961agacagcttg cagtgcacct gtgaggagat gaacgacatc aacgcgccct atctggtcat 1021gggacagaaa cagggtgggg agctggtgat cacctcggtg aagcggtggc agaaggggca 1081gagagagttc aagcgcatct cccgcagcat ccgcaagctg cagtgctagt cccggcatcc 1141tgatggctcc gacaggcctg ctccagagca cggctgacca tttctgctcc gggatctcag 1201ctcccgttcc ccaagcacac tcctagctgc tccagtctca gcctgggcag cttccccctg 1261ccttttgcac gtttgcatcc ccagcatttc ctgagttata aggccacagg agtggatagc 1321tgttttcacc taaaggaaaa gcccacccga atcttgtaga aatattcaaa ctaataaaat 1381catgaatatt tttatgaagt ttaaaaatag ctcactttaa agctagtttt gaataggtgc 1441aactgtgact tgggtctggt tggttgttgt ttgttgtttt gagtcagctg attttcactt 1501cccactgagg ttgtcataac atgcaaattg cttcaatttt ctctgtggcc caaacttgtg 1561ggtcacaaac cctgttgaga taaagctggc tgttatctca acatcttcat cagctccaga 1621ctgagactca gtgtctaagt cttacaacaa ttcatcattt tataccttca atgggaactt 1681aaactgttac atgtatcaca ttccagctac aatacttcca tttattagaa gcacattaac 1741catttctata gcatgatttc ttcaagtaaa aggcaaaaga tataaatttt ataattgact 1801tgagtacttt aagccttgtt taaaacattt cttacttaac ttttgcaaat taaacccatt 1861gtagcttacc tgtaatatac atagtagttt acctttaaaa gttgtaaaaa tattgcttta 1921accaacactg taaatatttc agataaacat tatattcttg tatataaact ttacatcctg 1981ttttacctat aaaaaaaaaa aaaaa Human SFRP2 protein sequence (SEQ ID NO: 7)MLQGPGSLLLLFLASHCCLGSARGLFLFGQPDFSYKRSNCKPIPANLQLCHGIEYQNMRLPNLLGHETMKEVLEQAGAWIPLVMKQCHPDTKKFLCSLFA PVCLDDLDETIQPCHSLCVQVKDRCAPVMSAFGFPWPDMLECDRFPQDNDLCIPLASSD HLLPATEEAPKVCEACKNKNDDDNDIMETLCKNDFALKIKVKEITYINRDTKIILETKSKTIYKLNGVSERDLKKSVLWLKDSLQCTCEEMNDINAPYLVMGQKQGGELVITSVKRW        QKGQREFKRISRSIRKLQC Human SFRP3 mRNA sequence (SEQ ID NO: 8) 1gttgggaaag agcagcctgg gcggcagggg cggtggctgg agctcggtaa agctcgtggg 61accccattgg gggaatttga tccaaggaag cggtgattgc cgggggagga gaagctccca 121gatccttgtg tccacttgca gcgggggagg cggagacggc ggagcgggcc ttttggcgtc 181cactgcgcgg ctgcaccctg ccccatcctg ccgggatcat ggtctgcggc agcccgggag 241ggatgctgct gctgcgggcc gggctgcttg ccctggctgc tctctgcctg ctccgggtgc 301ccggggctcg ggctgcagcc tgtgagcccg tccgcatccc cctgtgcaag tccctgccct 361ggaacatgac taagatgccc aaccacctgc accacagcac tcaggccaac gccatcctgg 421ccatcgagca gttcgaaggt ctgctgggca cccactgcag ccccgatctg ctcttcttcc 481tctgtgccat gtacgcgccc atctgcacca ttgacttcca gcacgagccc atcaagccct 541gtaagtctgt gtgcgagcgg gcccggcagg gctgtgagcc catactcatc aagtaccgcc 601actcgtggcc ggagaacctg gcctgcgagg agctgccagt gtacgacagg ggcgtgtgca 661tctctcccga ggccatcgtt actgcggacg gagctgattt tcctatggat tctagtaacg 721gaaactgtag aggggcaagc agtgaacgct gtaaatgtaa gcctattaga gctacacaga 781agacctattt ccggaacaat tacaactatg tcattcgggc taaagttaaa gagataaaga 841ctaagtgcca tgatgtgact gcagtagtgg aggtgaagga gattctaaag tcctctctgg 901taaacattcc acgggacact gtcaacctct ataccagctc tggctgcctc tgccctccac 961ttaatgttaa tgaggaatat atcatcatgg gctatgaaga tgaggaacgt tccagattac 1021tcttggtgga aggctctata gctgagaagt ggaaggatcg actcggtaaa aaagttaagc 1081gctgggatat gaagcttcgt catcttggac tcagtaaaag tgattctagc aatagtgatt 1141ccactcagag tcagaagtct ggcaggaact cgaacccccg gcaagcacgc aactaaatcc 1201cgaaatacaa aaagtaacac agtggacttc ctattaagac ttacttgcat tgctggacta 1261gcaaaggaaa attgcactat tgcacatcat attctattgt ttactataaa aatcatgtga 1321taactgatta ttacttctgt ttctcttttg gtttctgctt ctctcttctc tcaacccctt 1381tgtaatggtt tgggggcaga ctcttaagta tattgtgagt tttctatttc actaatcatg 1441agaaaaactg ttcttttgca ataataataa attaaacatg ctgttaccag agcctctttg 1501ctggagtctc cagatgttaa tttactttct gcaccccaat tgggaatgca atattggatg 1561aaaagagagg tttctggtat tcacagaaag ctagatatgc cttaaaacat actctgccga 1621tctaattaca gccttatttt tgtatgcctt ttgggcattc tcctcatgct tagaaagttc 1681caaatgttta taaaggtaaa atggcagttt gaagtcaaat gtcacatagg caaagcaatc 1741aagcaccagg aagtgtttat gaggaaacaa cacccaagat gaattatttt tgagactgtc 1801aggaagtaaa ataaatagga gcttaagaaa gaacattttg cctgattgag aagcacaact 1861gaaaccagta gccgctgggg tgttaatggt agcattcttc ttttggcaat acatttgatt 1921tgttcatgaa tatattaatc agcattagag aaatgaatta taactagaca tctgctgtta 1981tcaccatagt tttgtttaat ttgcttcctt ttaaataaac ccattggtga aagtcccaaa 2041aaaaaaaaaa aaaaaaaa Human SFRP3 protein sequence (SEQ ID NO: 9)MVCGSPGGMLLLRAGLLALAALCLLRVPGARAAACEPVRIPLCKSLPWNMTKMPNHLHHSTQANAILAIEQFEGLLGTHCSPDLLFFLCAMYAPICTIDFQHEPIKPCKSVCERARQGCEPILIKYRHSWPENLACEELPVYDRGVCISPEAIVTADGADFPMDSSNGNCRGASSERCKCKPIRATQKTYFRNNYNYVIRAKVKEIKTKCHDVTAV VEVKEILKSSLVNIPRDTVNLYTSSGCLCPPLNVNEEYIIMGYEDEERSRLLLVEGSIAEKWKDRLGKKVKRWDMKLRHLGLSKSDSSNSDSTQSQKSGRNSNPRQARN

Coronary Disorders

Many patients are either at risk for or have suffered from various typesof heart failure, including myocardial infarction, symptomatic orunsymptomatic left ventricular dysfunction, or congestive heart failure(CHF). An estimated 4.9 million Americans are now diagnosed with CHF,with 400,000 new cases added annually. This year over 300,000 Americanswill die from congestive heart failure. Without therapeutic invention,cardiac muscle does not normally have reparative potential. The abilityto augment weakened cardiac muscle as described herein is a majoradvance in the treatment of cardiomyopathy and heart failure. Despiteadvances in the medical therapy of heart failure, the mortality due tothis disorder remains high, where most patients die within one to fiveyears after diagnosis.

Coronary disorders are categorized into at least two groups. Acutecoronary disorders include myocardial infarction, and chronic coronarydisorders include chronic coronary ischemia, arteriosclerosis,congestive heart failure, angina, atherosclerosis, and myocardialhypertrophy. Other coronary disorders include stroke, myocardialinfarction, dilated cardiomyopathy, restenosis, coronary artery disease,heart failure, arrhythmia, angina, or hypertension.

Acute coronary disorders result in a sudden blockage of the blood supplyto the heart which deprives the heart tissue of oxygen and nutrients,resulting in damage and death of the cardiac tissue. In contrast,chronic coronary disorders are characterized by a gradual decrease ofoxygen and blood supply to the heart tissue overtime causing progressivedamage and the eventual death of cardiac tissue.

Cytoprotective Compounds

A cytoprotective (i.e., cell protective or regenerative) compound is acompound that that is capable of inhibiting cell damage such asapoptosis induced or oxidative-stress induced cell death. Cytoprotectivecompounds also include compounds that induce cell repair andregeneration. A cytoprotective compound is a polypeptide or nucleic acidencoding the polypeptide, the expression of which is increased inMSC-Akt cells under hypoxic conditions as compared to normoxiccondition. For example, a cytoprotective polypeptide includes Sfrp-1, 2,and/or 3, adipsin, adrenomedullin, chemokine (C—C motif) ligand 2,cysteine rich protein 61, lysyl oxidase-like 2, serine proteinaseinhibitor or vascular endothelial growth factor or fragment thereof.Other proteins/polypeptides with cytoprotective and regenerativeproperties include h1, 5, 8, 12, and 13. In some aspects the compound isa nucleic acid that increases expression of a nucleic acid that encodesa polypeptide or an agonist of a cytoprotective polypeptide.

Therapeutic Methods

The invention provides methods of inhibiting cell or tissue damage andischemic or reperfusion related injuries. Also included are methods ofregenerating injured myocardial tissue. The therapeutic methods includeadministering to a subject, or contacting a cell or tissue with acomposition containing a cytoprotective compound.

Cell/tissue damage is characterized by a loss of one or more cellularfunctions characteristic of the cell type which can lead to eventualcell death. For example, cell damage to a cardiomyocyte results in theloss contractile function of the cell resulting in a loss of ventricularfunction of the heart tissue. An ischemic or reperfusion related injuryresults in tissue necrosis and scar formation.

Injured myocardial tissue is defined for example by necrosis, scarringor yellow softening of the myocardial tissue. Injured myocardial tissueleads to one or more of several mechanical complications of the heart,such as ventricular dysfunction, decrease forward cardiac output, aswell as inflammation of the lining around the heart (i.e.,pericarditis). Accordingly, regenerating injured myocardial tissueresults in histological and functional restoration of the tissue.

The cell is any cell subject to apoptotic or oxidative stress inducedcell death. For example, the cell is a cardiac cell such as acardiomyocyte, a liver cell or a kidney cell. Tissues to be treatedinclude a cardiac tissue, a pulmonary tissue, or a hepatic tissue. Forexample, the tissue is an muscle tissue such as heart muscle. The tissuehas been damaged by disease or deprivation of oxygen.

Cells or tissues are directly contacted with a cytoprotective compound,e.g. by direct injection into the myocardium. Alternatively, thecytoprotective compound is administered systemically. The cytoprotectivecompounds are administered in an amount sufficient to decrease (e.g.,inhibit) apoptosis induced or oxidative stress induced cell death ascompared to untreated cells or tissues. Cells undergoing apoptosis areidentified by detecting cell shrinkage, membrane blebbing, caspaseactivation, chromatin condensation and fragmentation as is well know inthe art. Cell undergoing oxidative stress are identified by detecting anincrease production of reactive oxygen species (ROS). A decrease in celldeath (i.e., an increase in cell viability) is measured by usingstandard cell viability measurements such as BrdU incorporation assayand trypan blue exclusion.

The methods are useful to alleviate the symptoms of a variety disorders,such as disorders associated with aberrant cell damage, ischemicdisorders, and reperfusion related disorders. For example, the methodsare useful in alleviating a symptom of stroke, myocardial infarction,chronic coronary ischemia, arteriosclerosis, congestive heart failure,dilated cardiomyopathy, restenosis, coronary artery disease, heartfailure, arrhythmia, angina, atherosclerosis, hypertension, renalfailure, kidney ischemia or myocardial hypertrophy. The disorders arediagnosed and or monitored, typically by a physician using standardmethodologies. Alleviation of one or more symptoms of the disorderindicates that the compound confers a clinical benefit, such as areduction in one or more of the following symptoms: shortness of breath,fluid retention, headaches, dizzy spells, chest pain, left shoulder orarm pain, and ventricular dysfunction

Therapeutic Administration

The invention includes administering to a subject a compositioncomprising a cytoprotective compound (also referred to herein as a“therapeutic compound”).

An effective amount of a therapeutic compound administered systemicallyin the range of about 0.1 mg/kg to about 150 mg/kg. Proteins or peptidesare administered directly into the heart by injection at a dose of1-1000 μg. For example, 10, 20, 30, 40, 50, 60, 75, 100 μg areadministered by myocardial injection. Effective doses vary, asrecognized by those skilled in the art, depending on route ofadministration, excipient usage, and coadministration with othertherapeutic treatments including use of other anti-apoptotic agents ortherapeutic agents for treating, preventing or alleviating a symptom ofa particular cardiac disorder. A therapeutic regimen is carried out byidentifying a mammal, e.g., a human patient suffering from (or at riskof developing) an cardiac disorder, using standard methods.

The pharmaceutical compound is administered to such an individual usingmethods known in the art. Preferably, the compound is administeredorally, nasally, topically or parenterally, e.g., subcutaneously,intraperitoneally, intramuscularly, and intravenously. The compound isadministered prophylactically, or after the detection of an cardiacevent such as a heart attack. The compound is optionally formulated as acomponent of a cocktail of therapeutic drugs to treat cardiac disorders.Examples of formulations suitable for parenteral administration includeaqueous solutions of the active agent in an isotonic saline solution, a5% glucose solution, or another standard pharmaceutically acceptableexcipient. Standard solubilizing agents such as PVP or cyclodextrins arealso utilized as pharmaceutical excipients for delivery of thetherapeutic compounds.

The therapeutic compounds described herein are formulated intocompositions for administration utilizing conventional methods. Forexample, cytoprotective compounds are formulated in a capsule or atablet for oral administration. Capsules may contain any standardpharmaceutically acceptable materials such as gelatin or cellulose.Tablets are formulated in accordance with conventional procedures bycompressing mixtures of a therapeutic compound with a solid carrier anda lubricant. Examples of solid carriers include starch and sugarbentonite. The compound is administered in the form of a hard shelltablet or a capsule containing a binder, e.g., lactose or mannitol, aconventional filler, and a tableting agent. Other formulations includean ointment, suppository, paste, spray, patch, cream, gel, resorbablesponge, or foam. Such formulations are produced using methods well knownin the art.

Cytoprotective compounds are effective upon direct contact of thecompound with the affected tissue, e.g. heart muscle. Alternatively,cytoprotective compounds are administered systemically. Additionally,compounds are administered by implanting (either directly into an organsuch as the heart or subcutaneously) a solid or resorbable matrix whichslowly releases the compound into adjacent and surrounding tissues ofthe subject. For example, the compound is delivered to the cardiactissue (i.e., myocardium, pericardium, or endocardium) by directintracoronary injection through the chest wall or using standardpercutaneous catheter based methods under fluoroscopic guidance fordirect injection into tissue such as the myocardium or infusion of aninhibitor from a stent or catheter which is inserted into a bodilylumen. Any variety of coronary catheter, or a perfusion catheter, isused to administer the compound. Alternatively, the compound is coatedor impregnated on a stent that is placed in a coronary vessel.

The present invention is further illustrated, but not limited, by thefollowing examples.

Example 1 The Family of Secreted Frizzled Related Proteins MediateAkt-MSC Cardiac Protection and Repair Through Paracrine Mechanisms

Loss of myocardial tissue due to ischemia and infarction usually leadsto inflammation, scarring and cardiac myocyte hypertrophy. However,since the myocardium has limited endogenous repair and regenerativecapacity, these compensatory pathophysiological responses to myocardialdamage are frequently inefficient to sustain cardiac function, resultingeventually in cardiac dilation and failure.

Cellular cardiomyoplasty has been proposed as a potential approach forreconstitution of infarcted myocardium and recuperation of cardiacfunction. Several cell-based strategies have evolved using a variety ofalternatives, such as skeletal muscle myoblasts, embryonic stem cells,fetal cardiomyocytes, myocardial stem cells and marrow-derivedmesenchymal stem cells (MSC). Among these methods, the use of MSCs hasshown much promise for clinical applications.

Protection may result from differentiation of donor cells intocardiomyocytes, fusion of donor cells with host cardiomyocytes, orthrough enhanced myocardial perfusion. A significant mechanism by whichcardiomyocyte survival/function is mediated by stem cells is throughparacrine effects.

Intramyocardial transplantation of MSCs overexpressing the survival geneAkt (Akt-MSCs) resulted in reduced infarct size and volume, ventricularremodeling and cardiac dysfunction, 2 weeks after infarction, whencompared to hearts transplanted with control MSCs alone. Moreover,conditioned medium from Akt-MSCs provided cytoprotection of cardiacmyocytes exposed to hypoxia in vitro and once injected into infarctedhearts to dramatically limited the infarct size and preventedventricular dysfunction within 72 hours. Since this early effect cannotbe readily explained by significant regeneration of cardiac myocytesfrom the donor cells or enhancement of angiogenesis, these data indicatethat the observed effect is due to paracrine factors released by theAkt-MSCs that prevent myocyte loss.

Although it has been reported that native MSCs can secrete angiogenicfactors and cytokines, the ability of bone marrow derived MSCs,especially Akt-MSCs, to produce factor(s) capable of acutely protectingthe cardiomyocytes from cell death has not been previously documented.Given that apoptosis is the principal cause of myocytes loss in theacute phase of MI, therapeutic methods that prevent or reduce apoptoticcell death are effective in reducing the severity and extent ofmyocardial infarction. Paracrine factor(s) secreted by MSCs wereidentified, and biological evidence of their therapeutic potential isdescribed below.

A strategy was developed that allows large-scale identification andfunctional screening of secreted factors that are responsible for theenhanced cytoprotective effect of the Akt-MSCs. First, microarrayanalysis of Akt-MSC and control MSC under normoxia and 6 h of hypoxiawas performed. Approximately 62 transcripts that were differentiallyregulated between the Akt-MSC and control MSC under normoxia or hypoxiaencode for known secreted proteins based on their annotation. Includedin this list were three members of the secreted Frizzled-related protein(Sfrp) family, Sfr1, Sfrp2 and Sfrp3. Sfrps bind to Wnt ligands or theirfrizzled receptors and modulate Wnt signaling. All three factors areassociated with regulation of cell fate, differentiation, and cell deathand cell growth. The data described herein provide evidence thatAkt-MSCs exert an early protection to the injured heart by secretingSfrps, which then modulate the X pathway in cardiac myocytes to preventcell death.

The following material and methods were used to generate the datadescribed below.

Purification of Mesenchymal Stem Cells and Retroviral Transduction.

Bone marrow cells from 8-10 week-old wild type male C57BL/6J mice(Jackson Laboratory), were collected in a-modified minimum essentialmedia supplemented with 17% fetal bovine serum; 100 units/ml penicillin,100 mg/ml streptomycin; amphotericin B 0.25 mg/ml. Mononuclear cellswere then isolated from aspirates by Ficoll-Paque (Amersham Biosciences)gradient centrifugation. For the retroviral transduction, murine Akt1cDNA tagged with a c-myc epitope by PCR-amplification was cloned intopMSCV-IRES-GFP vector. To overexpress Akt/GFP (Akt-MSC) or GFP alone(GFP-MSC), MSCs were infected with high-titer VSV-G pseudotypedretrovirus.

Gene Expression Profiling and RNA Validation

Eight micrograms of total RNA from mouse GFP-MSCs and Akt-MSCs (n=3 pergroup) under normoxia or hypoxia (6 hours) were used for microarrayanalysis. Affymetrix GeneChips of Mouse Genome 430 2.0 Arrays(Affymetrix. CA), which allows analysis of ˜45000 transcrips, wasperformed in triplicate, and analyzed with Affymetrix Microarray Suite(MAS 5.0). For further analysis various Dhip was used. All possiblecomparisons (Akt-MSC normoxia vs. GFP-MSC normoxia, Akt-MSC hypoxia vs.GFP-MSC hypoxia, GFP-MSC hypoxia vs. GFP-MSC normoxia and Akt-MSChypoxia vs. Akt-MSC normoxia) were tested. The transcripts were thenannotated using various databases to compile a list of potent secretedcandidates.

Gene expression profiling was determined by quantitative real-timeRT-PCR (QPCR) for selected genes with appropriate primer mixtures(TaqMan® Gene Expression Assays, No. 4331182) from Applied Biosystems(Sfrp1, Mm00489161; Sfrp2, Mm00485986; Sfrp3(Frzb), Mm00441378; Gapdh,Mm99999915).

Conditioned Media Collection and Concentration

Passage 3 to 5 GFP-MSCs and Akt-MSCs reached to 90% confluence in 10 cmdishes. The cells were then left either into a standard incubator or thehypoxic chamber in the medium (αMEM+0.2% FBS+ITS) for 6 hours. Plateswith medium only were also left at the same conditions ascontrol-conditioned medium. The medium was concentrated up to 50× usinga Millipore system with membrane (Amicon Ultra-15).

Western Blotting

Proteins from conditioned medium from MSCs were separated by SDS pagegel (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad).The blots were incubated with Sfrp2 primary antibody (Santa CruzBiotechnology, Inc.) and then with appropriate secondary antibodyconjugated with horseradish peroxidase (Amersham Biosciences). Complexeswere detected by chemiluminescence (LumiGLO, Cell Signaling).

Suppression of Secreted Factor Effect by siRNA

GFP-MSCs or Akt-MSCs were incubated overnight with OptiMEM mediumcontaining 1 μM siRNA for Sfrps (Sfrp1, sense (5′→3′):CGGAUUGUAAAGAACUGCATT (SEQ ID NO:10), antisens (5′→3′):UGCAGUUCUUUACAAUCCGTT (SEQ ID NO:11); Sfrp2, sense (5′→3′):GGACGACAACGACAUCAUGTT (SEQ ID NO:12), antisense (5′→3′):CAUGAUGUCGUUGUCGUCCTC (SEQ ID NO:13); Sfrp3, sense (5′→3′):CCGUCAAUCUUUAUACCACTT (SEQ ID NO:14), antisense (5′→3′):GUGGUAUAAAGAUUGACGGTG (SEQ ID NO:15); Ambion). Rhodamine-labeled GFPsiRNA (Qiagen) was used to assess the efficiency of transfection. Cellswere incubated in normal medium for 48 hours, then exposed to a serumfree medium (αMEM+0.2% FBS+ITS) at normoxia or hypoxia as describedabove. The medium was concentrated for further analysis. The efficiencyof the siRNA-mediated reduction of Sfrps was assessed by QPCR using 18Sas a control.

Adult Rat Ventricular Myocyte Isolation and Quantification of ApoptoticCardiomyocytes

Adult rat ventricular myocytes (ARVMs) were isolated by enzymaticdissociation. 1×10⁶ cells were incubated in 10 cm dishes (BectonDickinson) overnight with full 199 medium (0.2% albumin, 2 mM carnitine,5 mM creatine, 5 mM taurine and 1 μg/ml of recombinant human insulin,Sigma). On the following day, the medium was replaced with optimalmedium according to different assays. Hypoxic condition was created byincubating the cells at 37° C. into a hypoxia chamber with an atmosphereof 5% CO₂/95% N₂. Oxygen level into the chamber was controlled to 0.5%.

Apoptosis was determined by measuring the activity of cleaved-caspase 3using a caspase-specific fluorogenic substrate according to the protocolfor Caspase 3 assay kit (Sigma, St. Louis, Mo.). ARVMs were lysed aftertreatment with SFRPs for 24 hours under hypoxia. 5 ul of cell extractwas incubated in reaction buffer at room temperature for 1 hour. Theenzyme-catalyzed release of 7-amino-4-methyl coumarin (AMC) was measuredby a fluorescence microplate reader. Fluorescent units were converted topmole AMC/μl sample/min/μg protein, using a standard curve of AMC.

Quantitation of Morphologic Changes of ARVC Following Hypoxic Exposure

Isolated cardiomyocytes were seeded in multi-well plates (BectonDickinson, Franklin Lakes, N.J., USA) precoated with laminin (1 μg/cm²)and left overnight in standard growth medium (M199). One day later, themedium was replaced by serum-free medium with different doses of Sfrp2.The ARVCs were then placed in the hypoxia chamber. The viability of theARVCs was evaluated on the basis of their morphology using a phasecontrast microscope, and rod-shaped cardiomyocytes were consideredviable. The number of round shaped cardiomyocytes was counted in 6random high power fields and expressed as a percentage of total numberof cells.

Myocardial Infarction Model and Determination of Infarct Size

Ligation of the left anterior descending coronary artery was performedon 170 to 200 grams female Sprague Dawley rats (Harlan WorldHeadquarters, Indianapolis, Ind.). A left thoracotomy was performedunder anesthesia, and the vessel was ligated with a silk suture atmidway between the left atrium and the apex of the heart. The infarctionwas then assessed by the change of color and kinesis of the apex and theanterior-lateral wall. Thirty minutes later 250 μl conditioned media wasinjected in 5 different sites around the border zone. An equivalentamount of PBS was injected in the control group. Then the wound wassutured immediately.

Infarct size was analyzed by staining the tissue 5 min at 37° C. withplanar morphometry in triphenyl tetrazolium chloride (TTC, SigmaChemicals) followed by fixation of 12 hours in 10% phosphate-bufferedformalin, and expressed as a percentage of the total ventricular area.

Akt Regulated Expression of Sfrps in MSCs

Since the secreted frizzled-related sequence, protein 2 (Sfrp2) appearedto be expressed highly only in Akt-MSCs, and two other members of thesame family (Sfrp1 and Sfrp3) were also upregulated in these cells,efforts were focused on these molecules. First, MSCs-Akt and controlGfp-MSCs were cultured under normoxia or 6 hours of hypoxia and the RNAwas collected and used to confirm the microarray data by quantitativePCR (Q-PCR). The expression pattern of all genes Sfrp1, Sfrp2 and Sfrp3was consistent with the microarray results. Although both Sfrp1 andSfrp3 exhibited a consistent trend (P<0.1) of being expressed higher inAkt-MSCs, the most dramatic and significant differences were shown inthe Sfrp2 levels (almost undetectable in control cells as opposed tohigh levels in Akt-MSCs). No significant changes were observed in thelevels of all three genes in regard to hypoxia treatment in eithercontrol MSC or Akt-MSCs.

To further validate the observations at the protein level and toevaluate the effect of Akt on Sfrp2 expression, control mouse MSCs andAkt-MSCs were cultured under normoxic or hypoxic conditions for 6 hourswith PI3K inhibitor (LY294002 50 mM) or vehicle. The conditioned mediumwas then collected and concentrated for protein analysis. Sfrp2 washighly expressed in the conditioned medium from the Akt-MSC cells atboth normoxia and hypoxia. The levels of Sfrp2 were low or undetectablein the supernatant from GFP-MSCs under normoxia or hypoxia. Furthermore,the expression of Sfrp2 in the Akt-MSC cells was dependent to the PI3Kpathway since inhibition of the PI3K, also abolished Sfrp2 expressionfrom the medium. Sfrp1 and Sfrp3 showed similar patterns of proteinexpression.

Akt-MSCs Promote Cardiac Myocyte Cell Survival after Injury Through SfrpMediated Paracrine Effects

To determine whether Sfrps are a key mediator of the earlycytoprotective effect of the conditioned medium in vitro, the effects ofconditioned medium from cultured Akt-MSCs (Akt CM) and Akt-MSCs that didnot express Sfrp1, Sfrp2 or Sfrp3 due to siRNA treatment (Akt-Sfrp2 CM)on the viability of adult rat cardiac myocytes (ARVCs) subjected tohypoxia were assessed. The conditioned media (CM) from Akt-MSCs ofGfp-MSCs was collected and concentrated after 6 hours of exposure toeither normoxia or hypoxia. The CM was then added to ARVCs that wereexposed to 24 h of hypoxia. The experimental conditions included ARVCsthat were incubated with either control conditioned medium (Ctr CM),conditioned medium from Akt-MSCs or Gfp-MSCs (Akt CM and Gfp CM) andconditioned medium Akt-MSCs or Gfp-MSCs that did not express Sfrp2 dueto siRNA treatment (Akt minus Sfrp2 CM and Gfp minus Sfrp2 CMrespectively). The data showed that ARVCs maintained under normoxicconditions for 24 hours were viable and exhibited their typicalrod-shaped appearance. Exposure of ARVCs to 24 hours of hypoxia incontrol conditioned medium (Ctr CM) resulted in a 200% increase in celldeath as indicated by caspase activity assay. Moreover, as expected theaddition of Gfp CM had no effect whereas addition of Akt CM resulted ina reduction of caspase activity (64% as compared to Ctr CM) to levelssimilar to normoxic conditions. However, exposure of hypoxic myocytes toAkt minus Sfrp2 CM resulted in increased caspase activity compared toCtr CM indicating higher cell death levels. Finally, reduction of Sfrp2expression in the Gfp CM did not have did not have any significantimpact on its effect on hypoxic cardiac myocytes.

To examine the direct effect of Sfrps on ARVCs we also performed gain offunction experiments in vitro. ARVCs were maintained in standard growthmedium at normoxia or at 24 h hypoxia. Sfrp1, Sfrp2, Sfrp3 or vehiclewas then added at various concentrations and apoptosis levels wereassessed as before by measuring caspase activity. Treatment with as lowas 0.1 ng/ml of Sfrp1 or Sfrp2 resulted in significant reduction incaspase activity (36% and 33% respectively). However, higherconcentrations of Sfrp1 showed reduced protection. On the contrary,Sfrp2 mediated reduction of cell death was positive correlated to higherconcentrations of the molecule and seemed to plateau around theconcentration of 10 ng/ml (55% reduction in caspase activity). Sfrp3treatment reduced caspase activity only in concentrations higher than 10ng/ml and overall was less potent that the other molecules (54%reduction at 500 ng/ml).

Finally, to corroborate the results from the apoptosis assays, therelative number of healthy ARVCs after 24 hours of hypoxia was assessedbased on their ATP synthesis levels. For this, again the cells weregrown in normoxia or hypoxia with PBS or Akt CM, Akt-Sfrp2 CM, 10 ng/mlSfrp2, or 500 ng/ml Sfrp3 for 24 h. Exposure of ARVCs to 24 h plus AktCM increased cell viability by 35% whereas medium from Akt cells thatdid not express Sfrp2 increased cell viability only by 9%. Treatmentwith Sfrp2 and Sfrp3 resulted in 24% and 17% increase in viabilityrespectively.

Sfrp Treatment Protects the Heart from Myocardial Injury

To elucidate the therapeutic potential of the Sfrps, we studied thedirect effects of Sfrps on infarct size by intramyocardial injection ofSfrp1, Sfrp2 or Sfrp3 peptide. For this, 1 μg of Sfrp1, Sfrp2 or Sfrp3were injected into 5 different sites in the heart at the infarct borderzone 30 minutes after coronary artery occlusion. Additional groupsincluded hearts injected with PBS as negative control, hearts injectedwith Akt CM as positive control and hearts injected with Akt minus Sfrp2CM to provide further evidence of Sfrp2 role in the protective Akt-MSCCM mediated cardiac protection in vivo. Hearts were isolated 72 hourslater and infarct size was estimated by TTC staining. Injection of Sfrp2had an effect of 69% reduction of infarct size, while injection of thesame concentration of Sfrp1 resulted in 50% reduction in infarct sizeand the same dose of Sfrp3 did not have any effect on infarct size.Since Sfrp2 have been shown to have the most potent effect from all thethree Sfrps tested, its physiological significance in Akt-MSC mediatedmyocardial protection in vivo was also evaluated. Injection of Akt CM ininfarcted hearts resulted in 71% reduction in the infarct size after MIwithin 72 hours, whereas injection Akt minus Sfrp2 CM did not show anysignificant protection. These results indicate that Sfrps secreted fromAkt-MSCs protects the myocardium from injury.

Sfrps Mediate Cardioprotection

Despite vigorous efforts and the great potential of cell-based therapiesfor cardiac disease, the mechanisms underlying their therapeutic effectare still under debate. The data described herein indicates that MSCsexert an early protective effect in the injured myocardium by preventingmyocyte cell death. This effect is enhanced by the overexpression of Aktand includes paracrine factors that regulate the Wnt signaling pathwayin cardiac myocytes. Sfrp modulators of Wnt pathway protect from hypoxiacell death in vitro and result in reduction of infarct size in vivo.

Members of the Secreted frizzled-related protein (Sfrp) family act asmodulators of the Wnt signaling pathway thereby influencing a range ofbiological processes, such as cell fate, cell adhesion, differentiationand survival. Sfrps are inhibitors of the Wnt signaling pathway. Theyact through binding of Wnts and altering their ability to bind to theirfrizzled receptors or by forming non functional complexes with thefrizzled receptors themselves. However, some studies suggest that Sfrp1at low concentrations may actually promote Wnt signaling. Furthermore,it has been reported that similar concentrations of Sfrp1 and Sfrp2result in different cellular responses. For instance, Sfrp1 has beenshown to sensitize cells to TNF induced apoptosis whereas Sfrp2conferred resistance. A proposed explanation for these observations isthat the Sfrp specific effects are closely dependent on the range oftheir Wnt partners present, the relative affinities of different Sfrpsfor Wnt or Frizzled receptors, tissues specific responses or biphasicresponses to different concentrations of Sfrp. The present data supportthis mechanis, since the three different Sfrps tested conferred variabledegrees of protection to cardiac myocytes and these effect was dependenton their concentration levels.

Prior to the invention, little was known about the role of Sfrps incardiac tissue. Sfrp1 has been associated with heart morphogeneiss,whereas Sfpr3 and Sfrp4 were found to be upregulated in volume overloadinduced hypertrophy. Evidence suggests that they are play a role duringcardiac ischemia but again their role is diverse and not fullyunderstood. For instance, overexpression of Sfrp1 seemed to protect theheart from injury in a model of coronary ligation but has been reportedto alleviate it and reverse the benefit of preconditioning in a model ofischemia/reperfusion. Similarly few studies have been conducted inregard to the role of Wnt signaling in cardiac myocyte survival. Thepresent data provides evidence that Sfrp activates/inhibits Wntsignaling.

The data do not exclude additional paracrine effects from other proteinssecreted by the Akt-MSCs. Indeed, other secreted molecules are alsoexpressed and are involved in different aspects of cardiac repair suchas immunological responses, angiogenesis, recruitment/expansion ofcardiac stem cells, regeneration and/or remodeling. For example,administration of vascular endothelial growth factor A (a growth factorwith higher levels in Akt-MSCs) resulted in repaired myocardium bypromoting angiogenesis and vascularization. Moreover, paracrine factorsexert not only individual effects, but in some examples, one to factorenhances the effect of another, i.e, a synergistic relationship ispresent between the different secreted factors expressed by the MSCs. Inother examples, the presence of one factor inhibits the effects of oneor more others.

Paracrine factors, e.g., Sfrps, contained in conditioned medium fromAkt-MSCs are useful in therapeutic methods to prevent or reduce celldeath, e.g., apoptotic cell death, of cardiac cells. The data indicatesthat simple administration of Sfrp2 alone or in combination with othermolecules achieve cardioprotective results similar and in some casesbetter than those seen with stem cell based therapy. Methods that employthese paracrine factors have numerous advantages over cell basedtherapies. For example, many of the difficulties of stem cell basedtherapy such as availability of cells, laborious maintenance of celllines, limited alternative administration methods as well asdifficulties in successful delivery and survival of the cells can beavoided. Administration of a peptide or a cocktail of peptides to theinjured myocardium is a simpler, delivery methods, and dosages are moreeasily modified to achieve higher efficiency with lower toxicity or sideeffects and does not involve any of the ethical concerns associated withcell therapy.

Example 2 Secreted Frizzled Related Protein 2 is the Key Stem CellParacrine Factor Mediating Myocardial Survival and Repair

Using a comprehensive functional genomic strategy, Sfrp2 was shown to bea key stem cell paracrine factor that mediates myocardial survival andrepair following ischemic injury. Sfrp2 modulates Wnt signaling, andcardiomyocytes treated with secreted frizzled related protein increasecellular β-catenin and up-regulate expression of anti-apoptotic genes.These findings demonstrate the key role played by Sfrp2 in mediating theparacrine effects of Akt mesenchymal stem cells on tissue repair andidentify modulation of Wnt signaling as a strategy for treating heartdisease.

Microarray data confirmed by Western blot analysis demonstrated that oneof the most prominently expressed and secreted protein by Akt-MSCcompared to native MSC is the Sfrp2. Quantitative PCR showed 100 fold upregulation of Sfrp2 mRNA in Akt-MSC compared to control MSC. Sfrps aresecreted glycoprotein molecules that structurally resemble cell surfacefrizzled receptors but lack the transmembrane domain. They have beenincreasingly recognized as potent regulators of cellular Wnt signalingand have been implicated in diverse cellular processes such asregulation of cell fate, differentiation, proliferation and cell death.

Sfrp2 was found to play a major role in mediating the survival signal ofAkt-MSC on the ischemic myocardium. The data shows that Sfrp2 exertedsurvival effects on ischemic cardiomyocytes and that the pro-survivaleffects of Akt-MSC were markedly attenuated upon knockdown of Sfrp2using siRNA. Sfrp2 increased total cellular and nuclear β-catenin incardiomyocytes in vitro. Stabilization of β-catenin has beendemonstrated to protect neonatal rat cardiomyocytes againsthypoxia/re-oxygenation induced apoptosis. The canonical Wnt, Wnt3a, wasfound to be up-regulated in ischemic cardiomyocytes in vitro, and Wnt3ainduced apoptosis of cardiomyocytes. Sfrp2 blocked the pro-apoptoticeffect of Wnt3a. The data indicate that Sfrp2 is a major paracrinemediator of Akt-MSC myocardial survival and reparative effects andindicate that modulators of Wnt signaling such as Sfrp2 are useful astherapeutic agents in the management of myocardial injury.

Experiments were carried out as described above. Further profiling ofsecreted factors to identify cytoprotective proteins is described below.

Profiling of Secreted Factors Expressed in MSCs

To identify potential Akt-MSC secreted candidate paracrine factorsmediating myocardial cell survival following ischemic injury, AffymetrixGeneChip® Mouse Genome 430 2.0 Arrays, which allows analysis ofapproximately 45,000 transcripts was used. Expression levels and qualityanalysis were carried out with the Affymetrix MAS 5.0 software. Furtheranalysis was performed using the dChip software based on the followingfiltering criteria: a) Transcripts expressed (P call) in at least one ofthe sample compared, b) Fold change: at least 1.2×, (90% lower boundconfidence). Approximately 650 transcripts were differentially regulatedbetween the Akt-MSC and the GFP-MSC. Included in this list were 169transcripts with unassigned function. The set of 650 transcripts wasqueried for transcripts coding for secreted proteins. This analysisrevealed 62 transcripts encoding for 51 unique genes that contribute tothe paracrine effects of the MSC cells (Table 1).

TABLE 1 Fold change Fold change Akt Akt vs. Gfp at vs. Gfp at Probe setGene Title Gene Symbol normoxia hypoxia 1426858_at inhibin beta-B Inhbb−2.27 −4.34 1423635_at bone morphogenetic protein 2 Bmp2 −3.82 −3.191456404_at a disintegrin-like and Adamts5 −1.22 −3.08 metalloprotease(reprolysin type) with thrombospondin type 1 motif, 5 (aggrecanase-2)1426152_a_c kit ligand/stem cell factor Kitl −1.64 −2.78 1427760_s_atProliferin Plf −3.15 −2.61 1431056_a_at lipoprotein lipase Lpl −2 −2.581450658_at a disintegrin-like and Adamts5 −1.71 −2.21 metalloprotease(reprolysin type) with thrombospondin type 1 motif, 5 (aggrecanase-2)1449528_at c-fos induced growth factor Figf −2.27 −2.14 1438953_at c-fosinduced growth factor Figf −3.02 −2.09 1415904_at lipoprotein lipase Lpl−1.55 −2.08 1418450_at immunoglobulin superfamily Islr −1.55 −2.06containing leucine-rich repeat 1426951_at cysteine-rich motor neuron 1Crim1 −2.41 −2 1437218_at fibronectin 1 Fn1 −1.89 −1.97 1438954_x_atc-fos induced growth factor Figf −3.03 −1.96 1435603_at secreted proteinSST3 SST3 −1.12 −1.93 1422561_at a disintegrin-like and Adamts5 −1.14−1.91 metalloprotease (reprolysin type) with thrombospondin type 1motif, 5 (aggrecanase-2) 1418061_at latent transforming growth Ltbp2−2.66 −1.87 factor beta binding protein 2 1451243_at arginylaminopeptidase Rnpep −1.34 −1.86 (aminopeptidase B) 1460302_atthrombospondin 1 Thbs1 1.03 −1.84 1417234_at matrix metalloproteinase 11Mmp11 −1.59 −1.82 1438936_s_at Angiogenin Ang 1.18 −1.82 1447862_x_atthrombospondin 2 Thbs2 −1.33 −1.8 1425985_s_at mannan-binding lectinMasp1 −1.72 −1.79 serine protease 1 1448117_at kit ligand Kitl −1.23−1.79 1438937_x_at Angiogenin Ang −1.22 −1.76 1416164_at fibulin 5 Fbln5−1.35 −1.72 1448823_at chemokine (C-X-C motif) Cxcl12 −1.1 −1.62 ligand12 1415949_at carboxypeptidase E Cpe −1.33 −1.6 1416953_at connectivetissue growth Ctgf −6.01 −1.57 factor 1449187_at platelet derived growthPdgfa −2.33 −1.55 factor, alpha 1423396_at Angiotensinogen Agt −2.48−1.51 1421228_at chemokine (C-C motif) Ccl7 −3.4 −1.25 ligand 71438133_a_at cysteine rich protein 61 Cyr61 −3.93 −1.18 1419662_atOsteoglycin Ogn 2.19 −1.07 1420380_at chemokine (C-C motif) Ccl2 −6.731.01 ligand 2 1416039_x_at cysteine rich protein 61 Cyr61 −4.61 1.041417130_s_at angiopoietin-like 4 Angptl4 −1.04 1.02 1421991_a_atinsulin-like growth factor Igfbp4 2.32 1.19 binding protein 4 1416371_atapolipoprotein D Apod 1.88 1.34 1423294_at mesoderm specific Mest 2.211.34 transcript 1416594_at secreted frizzled-related Sfrp1 2.23 1.42sequence protein 1 1450325_at angiopoietin 4 Agpt4 2.43 1.6 1417634_atsuperoxide dismutase 3, Sod3 4.31 1.61 extracellular 1417256_at matrixmetalloproteinase 13 Mmp13 2.21 1.74 1417633_at superoxide dismutase 3,Sod3 3.23 1.78 extracellular 1429348_at sema domain, Sema3c 2.61 1.92immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin)3C 1451866_a_at hepatocyte growth factor Hgf 2.32 2.26 1429679_atleucine rich repeat Lrrc17 2.36 2.35 containing 17 1452436_at lysyloxidase-like 2 Loxl2 1.8 2.62 1431591_s_at interferon, alpha-inducibleG1p2 4.75 2.71 protein 1448424_at secreted frizzled-related Sfrp3 3.153.14 sequence protein 3 1419043_a_at interferon-inducible GTPase 1 Iigp13.97 3.15 1419042_at interferon-inducible GTPase 1 Iigp1 4.61 3.551451959_a_at vascular endothelial growth Vegfa −1.07 3.64 factor A1447839_x_at Adrenomedullin Adm −3.72 4.03 1417867_at Adipsin Adn 3.54.15 1448254_at Pleiotrophin Ptn 5.21 4.48 1416211_a_at Pleiotrophin Ptn5.68 4.79 1416077_at Adrenomedullin Adm −2.78 8.36 1419149_at serine (orcysteine) Serpine1 −6.34 10.35 proteinase inhibitor, clade E, member 11448201_at secreted frizzled-related Sfrp2 10.04 11.66 sequence protein2

Among these upregulated genes, Sfrp2 was the most dramaticallyupregulated. Other cytokines such as Vegf, Hgf and FGF were notdifferentially expressed between Akt-MSC and FP-MSC under normoxicconditions. The expression of Sfrp2 was Akt pathway dependent. Theexpression of the other Sfrp family members were minimally altered inAkt-MSC (FIG. 1A).

Akt Regulated Expression of Sfrps in MSCs

The results of microarray analysis was confirmed by quantitative PCR(Q-PCR). RNA was collected from cultured Akt-MSC and GFP-MSC that werecultured in vitro. As shown in FIG. 1B, the expression pattern of Sfrp1,Sfrp2 and Sfrp3 was consistent with the microarray results. NeitherSfrp1 and Sfrp3 was significantly upregulated in Akt-MSC vs GFP-MSC,whereas Sfrp2 expression was 100 fold higher in Akt-MSC.

To further validate our observations at the protein level and toevaluate the effect of Akt on Sfrp2 expression, control mouse MSCs andAkt-MSCs were cultured for 6 hours with PI3Kinase inhibitor (LY294002 50mM) or vehicle. The conditioned medium was then collected andconcentrated for Western blot protein analysis. As shown in FIG. 2A,Sfrp2 was secreted at high levels into the conditioned medium from theAkt-MSC cells (lanes 1). The levels of Sfrp2 were low or undetectable inthe conditioned medium of GFP-MSCs (lanes 2). Furthermore, theexpression/release of Sfrp2 in the Akt-MSC cells was dependent on thePI3K pathway since inhibition of the PI3K, abolished Sfrp2 accumulationin the medium (lanes 3).

Akt-MSCs Promote Cardiomyocyte Cell Survival Through ParacrineMechanisms Mediated by Sfrp

To prove whether Sfrp2 is a key paracrine mediator of the survivalsignaling of Akt-MSC, the apoptotic response (caspase activity) of adultrat ventricular cardiomyocytes (ARVC) exposed to conditioned mediumcollected from Akt-MSC treated with siRNA (Akt-MSC minus Sfrp2 CM)against Sfrp2 was evaluated. ARVC were subjected to hypoxia for 24 hoursin the presence of Akt-MSC CM, Akt-MSC minus Sfrp2 CM or standard growthmedium (GM). (FIGS. 2B,C). ARVCs maintained in standard growth mediumunder normoxic conditions for 24 hours were viable and exhibited theirtypical rod-shaped appearance while ARVC grown in the same medium andsubjected to 24 hour hypoxia exhibited a 82% increase in caspaseactivity (FIG. 2C). Compared to hypoxic ARVC maintained in standardgrowth medium, hypoxic ARVC exposed to Akt-MSC CM exhibited a 40%reduction in caspase activity (FIG. 3B). Moreover, exposure of hypoxiccardiomyocytes to Akt minus Sfrp2 CM resulted in a significant increasein caspase activity compared to hypoxic ARVC treated with Akt CM. A 33%increase in caspase activity was observed in hypoxic ARVC followingknockdown of Sfrp2 expression in Akt-MSC. These observations demonstratethe key role played by Sfrp2 in mediating survival effects of Akt-MSC CMon cardiomyocytes.

To examine the direct effect of Sfrp2 on ARVCs, gain of functionexperiments were carried out. ARVCs were maintained in standard growthmedium at normoxia or subjected to 24 h hypoxia. Sfrp2 or vehicle wasthen added at various concentrations and apoptosis levels were assessedby measuring caspase activity. Treatment with Sfrp2 resulted insignificant reduction in caspase activity, and a dose dependentcytoprotective response was observed with increasing Sfrp2concentrations up to 15 nM (FIG. 3A).

The cytoprotective effects of Sfrp2 on cardiomyocytes was confirmed byobserving changes in cardiomyocyte cell morphology following exposure tohypoxia. ARVC following exposure to 24 hour hypoxia, lose their typicalrod shaped morphology, become round in shape, subsequently detach anddie. Hypoxia alone increased the number of round shaped cardiomyocytesby approximately 36% (FIG. 3B, C). However when ARVC were treated withSfrp2 (3 nM), the number of round shaped cardiomyocytes was decreased byapproximately 31% compared to untreated controls (FIG. 3B, C). The datastrongly indicate that Sfrp2 promotes cardiomyocyte survival andprotects cardiomyocytes from hypoxic injury.

Suppression of Sfrp2 Expression in Akt-MSCs Reduces the ParacrineProtection of Myocardial Injury In Vivo.

Experiments were carried out to evaluate the physiological significanceof Sfrp2 in Akt-MSC mediated paracrine myocardial protection in vivo. Todemonstrate the importance of Sfrp2 as a key paracrine factor mediatingprosurvival effects of injected Akt-MSC, in vivo effects of conditionedmedium collected from Akt-MSC treated with siRNA against Sfrp2 werecompared with those of untreated Akt-MSC CM. Akt-MSC treated with siRNAagainst Sfrp2 had a 60% decrease in Sfrp2 mRNA expression following 48hours of exposure to siRNA (FIG. 2B). The conditioned medium either fromuntreated or siRNA treated cells was collected, concentrated and theninjected into 5 different sites at the infarct border zone 30 minutesafter coronary artery ligation (a standard model for MI). Hearts werethen isolated 72 hours later and infarct size was estimated by TTCstaining. The results were analyzed by an investigator blinded to thetreatment groups. As shown (FIGS. 4A, B) injection of Akt CM ininfarcted hearts resulted in 71% reduction in the infarct size after MIwithin 72 hours, whereas injection of conditioned medium from siRNAtreated Akt-MSC did not show any significant protection. Collectively,these results indicate that Sfrp2 possesses cell survival signalingproperties and mediates myocardial protective effects followingmyocardial infarction.

Sfrp2 Leads to Upregulation of βCatenin in Hypoxic Cardiomyocytes

Sfrp2 is an antagonist of Wnt signaling. Unlike Sfrp1 which canpotentiate Wnt signaling under certain conditions, Sfrp2 has not beenknown to activate Wnt signaling. However, evidence described hereinindicates that Sfrp2 increases total cellular as well as nuclear βcatenin mimicking canonical Wnt signaling. Using Western blotting, Sfrp2was found to induce a dose dependent increase in nuclear as well astotal cellular β catenin levels in cardiomyocytes exposed to hypoxia(FIG. 5C). Increased β catenin within cardiomyocytes is associated withincreased cellular protection against ischemic injury in vitro. Thesedata indicate that Sfrp2 promotes the survival of cardiomyocytes againsthypoxia induced apoptosis via potentiation of canonical signaling.Experiments were then carried out to determine if Wnts are up-regulatedin cardiomyocytes exposed to 24 hour hypoxia. The data indicated thatWnt3a was expressed at very low levels in normoxic cells but increasedin hypoxic cells (FIG. 5A). Cardiomyocytes were incubated both undernormoxia and hypoxia/reoxygenation with Wnt3a alone and in combinationwith Sfrp2. The data demonstrated that under normoxic conditions, ascompared to control cardiomyocytes Wnt3a treatment resulted in a modestincrease in caspase 3 activity which was attenuated by Sfrp2 treatment.Furthermore, under hypoxia/reoxygenation conditions, Wnt3a treatmentresulted in a significant increase in caspase activity which wasinhibited by the addition of Sfrp2 (FIG. 5B).

Sfrp2 Upregulates Expression of Anti-Apoptotic Gene Birc1b in HypoxicCardiomyocytes

To further investigate the molecular mechanism by which Sfrp2 protectscardiomyocytes from cell death, RNA from hypoxic cardiomyocytesfollowing Sfrp2 treatment (10 ng/mL) was collected and expression ofmultiple genes involved in cell survival/death pathways was determinedusing microarray analysis. Using an oligo GE Array for rat signaltransduction pathways, gene expression of 95 marker genes associatedwith 18 different signaling pathways was analyzed. In this analysis, 43genes showed differential expression between the Sfrp2 treated and thecontrol cardiomyocytes. Sfrp2 upregulated the expression of Birc1b, ananti-apoptotic gene belonging to the neuronal apoptosis inhibitoryprotein (NAIP) family. Expression of other cytoprotective genes such asBcl2 were only minimally increased in hypoxic cardiomyocytes in thepresence of Sfrp2 (FIGS. 6A, B).

Sfrp-Based Therapy for Cardiac Disorders

Sfrp2 was identified as an Akt-MSC secreted protein exerting prosurvivaleffects on the myocardium. Several lines of evidence support the role ofSfrp2 as a principal mediator of anti apoptotic effects exerted on themyocardium by Akt-MSC. First, Sfrp2 expression is dramaticallyupregulated (100×) in Akt-MSC compared to GFP-MSC and itsexpression/secretion is dependent on the PI3 kinase/Akt pathway.Secondly, Sfrp2 conferred prosurvival effects on hypoxic cardiomyocytes.Moreover, knockdown of Sfrp2 expression resulted in the attenuation ofthe prosurvival action of Akt-MSC conditioned medium both in vitro andin vivo.

Sfrp2 is a secreted glycoprotein molecule that structurally resemblescell surface Frizzled receptors but lacks the latter's transmembranedomains. Sfrps compete with the frizzled receptor for Wnt ligands bydirect binding of Wnts thus preventing activation of Wnt signaling inthe cell. The Wnt family currently comprises 19 different proteins. Wntsare known to regulate organogenesis during embryonic development and inmammals and in other species such as amphibians and birds have beenimplicated in cardiac morphogenesis as well. They regulate diversecellular processes such as proliferation, differentiation and apoptosis,but the role of the Wnts in regulating such processes in the post natalheart was not known. Although various Wnts such as Wnt10b and severalfrizzled receptors are expressed in the human heart, it was not knownwhether they play a role in cardiac homeostasis. The data describedherein indicates that Sfrp2, a known modulator of Wnt signaling exertsprosurvival action on cardiomyocytes. The data demonstrate that Sfrp2increases as well as nuclear βcatenin within the hypoxic cardiomyocytein a dose dependent manner. βcatenin when activated translocates to thenucleus and initiates transcription of a wide variety of genes; thus thenuclear fraction represents a more accurate measure of activatedβcatenin. Sfrp1 has previously been shown to potentiate Wnt signaling bydirectly binding to Frizzled receptors. In hypoxic cardiomyocytes, Sfrp2binds locally present Wnts and alters the balance of intracellular Wntsignaling within a cardiomyocyte to favor a canonical pathway. Wnt3a wasfound to be upregulated in hypoxic cardiomyocytes. Wnt3a increasescardiomyocyte apoptosis and Sfrp2 blocks this effect of Wnt3a. Sfrp2 mayalso bind directly to frizzled receptor on cardiomyocytes activating thecanonical pathway. The data indicate that Sfrp2 by increasing cellularand nuclear βcatenin enhances the survival response of cardiomyocytesagainst hypoxia induced apoptosis. Sfrp2 also upregulated expression ofBirc1b, an anti-apoptotic gene belonging to the NAIP family. Sfrp2mediated increased βcatenin activates transcription of anti-apoptoticgenes such as Birc1b in hypoxic cardiomyocytes. Indeed, pharmacologicinhibition of GSK3β, resulting in increased βcatenin has been found toupregulate expression of anti-apoptotic genes such as Bcl2. Sfrp2 isinvolved in regulating cardiomyocyte cell survival and preservingcardiac function following myocardial infarction. Sfrp2 also plays arole as an important paracrine factor mediating beneficial effects ofstem cell therapy. Sfrp2 alters the local milieu around the infarct zoneto favor cardiomyocyte cell survival. Simple administration of Sfrp2protein or fragments that modulate the Wnt-βcatenin pathway achieveresults similar to stem cell based cardiac therapy, and a protein basedtherapy has advantages over cell based cardiac therapy for acutemyocardial infarction and other ischemic cardiac disorders.

Example 3 Sfrp2 Maintains Cells in a Stem Cell State

Sfrp2 was found to be strongly expressed by mouse embryonic stem cells(e.g., P19CL6 cell line which readily differentiates into cardiomyocytesunder certain conditions). Sfrp2 was found to strongly inhibitdifferentiation of the murine embryonic PI9C16 cell line. Overexpressionof Sfrp2 or addition of recombinant Sfrp2 protein inhibiteddifferentiation of these cells. This data indicates that Sfrp2, byinhibiting differentiation of stem cells and maintaining them in theundifferentiated state, plays a role in maintenance of a stem cellphenotype and self renewal of stem cells. When added to P19CL6 cells,purified Sfrp2 prevented these cells from differentiating intocardiomyocytes. This result indicates that Sfrp2 by inhibitingdifferentiation of embryonic stem cells and maintaining them in theundifferentiated state preserves a stem cell phenotype of such cells.Maintenance of stemness is a fundamental and essential property of stemcells. It is not only of essential biological importance but greatclinical significance. For example, bone marrow transplantation involvesselection and administration of hematopoietic stem cells. A composition,e.g., Sfrp2 or other paracrine factor, that maintain the stemness ofembryonic and adult stem cells is useful to preserve and maintain stemcells for tissue repair and regeneration.

Example 4 Identification of Protective Factors Secreted by Akt-MSC

Microarray analysis of Akt-MSC and control MSC under normoxia or hypoxiawas performed to identify transcripts that were differentially regulatedbetween these conditions. Using this approach, 61 proteins of knowparacrine function were identified, e.g., pleiotrophin, chemokineligands 2 and 7 and various angiogenic factors such as VEGFa,angiopoietin 4 and HGF. Upregulated transcripts with unassigned functionwere subjected to genomic analysis using a combination of bioinformaticsoftware programs that allows predictions of potential secretedpeptides. Putative secreted proteins thus identified were then screenedusing siRNA technologies in a high throughout cell-based assays toexamine key physiological mechanisms involved in the cardioprotectiveeffects of Akt-MSCs. Using this approach, secreted proteins wereidentified that are overexpressed in Akt-MSCs. One of these was highlyexpressed in Akt-MSCs but nearly undetectable in control MSCs. Permanentclones of Akt-siRNA knock down were then established for each of thesegenes and conditioned medium from these cells was compared toconditioned medium from Akt-MSCs for its cytoprotective effect incardiac myocytes in vitro by apoptosis and cell viability assays.

Subsequently, the open reading frames of these novel transcripts werecloned and expressed in E. coli as maltose binding protein (MBP) fusionproteins. Compared with MBP alone, one of the MBP-novel fusion proteins(Protein #12; “h12”) significantly reduced the H₂O₂-induced apoptosis inH9C2 myocytes. Protein 12 was re-cloned into pET vector to allow rapidpurification as a 6×His tagged recombinant protein. Since Protein 12 iscysteine rich, purification was performed under denaturing condition andthe protein was refolded by dialysis with a redox pair to promotedisulfide bond formation. To test the cardioprotective effects ofProtein 12, the effects of addition of this protein on H₂O₂-inducedapoptosis in H9C2 myocytes was evaluated. Myocytes were treated with 100μM H₂O₂ or vehicle and the levels of apoptosis was assessed by FACSanalysis following Annexin V/PI staining. H₂O₂ induced high levels ofearly apoptosis, yielding approximately 30% Annexin V positive cellswith less than 5% necrotic cells (PI positive). Pre-treatment of thecells with 10 nM of Protein 12 for 30 min reduced early apoptosis bynearly 50%. This protein significantly reduced H₂O₂ induced caspase 9activity in adult rat cardiomyocytes by 38.5%, dramatically inhibitedthe mitochondrial release of cytochrome C and increased the totalsurvival rate by 28%. The data indicate that this cysteine-rich Protein12 possesses cardio-protective effects of Akt-MSCs.

A total of 5 transcripts with previously undefined function were foundto account account for myocardial protection of AKT-MSCs. The openreading frame of these novel transcripts were subsequently cloned,expressed and purified from E. coli, as either fusion proteins ofmaltose binding protein-novel proteins or as 6×His tagged recombinantproteins. Protein No. 12, which is a cysteine-rich insoluble proteinwhen expressed in E. coli., was then purified under denaturing conditionand refolded by dialysis with a redox pair to promote disulfide bondformation. This No. 12 protein was used in various assays for oxidativestress induced apoptosis in cardiomyocytes and was found to have astrong cardio-protective effect.

For Human No. 12, the coding sequence without the predicted N-terminalsignal region (1158 base pairs) were amplified and cloned in-frame ofprotein translation into pMal-C vector to generate a fusion protein ofmaltose binding protein-Human No. 12, designated as MBP-h12 (˜80 KDa).Expression was induced by IPTG in E. coli. and purification of MBP-h12was done by standard affinity chromatography according to New EnglandBiolab's instructions. MBP-h12 was further purified by FPLC system.Compared with control MBP alone, this MBP-h12 fusion proteinsignificantly prevents H₂O₂-induced early apoptosis in H9C2 myocytes(˜30% reduction of apoptosis), by Annexin V/PI double staining with FACSanalysis. To gain further insight of protein No. 12′ function incardiovascular biology, same coding region (1158 base pairs) werere-amplified and cloned in-frame into pET 15b vector to generate6×His-tagged recombinant protein, designated as His-h12 (˜40 KDa).Protein was first purified under denaturing condition and refolded bydialysis with gradually decreasing amount of dithiothreital. Oxidizedand reduced of glutathione as the ‘redox pair’ was added in the finalstep to promote disulfide bond formation. Refolded His-h12 proteins weredialyzed extensively in PBS and were used in subsequent apoptosisassays.

FIG. 7 shows the results of Annexin V/PI staining with FACS analysis inH9C2 myocytes for early apoptosis. H9C2 myoctyes were seeded at 1×10⁴per well in 6-well plate one day before experiment. Recombinant His-h12proteins were added to the cells at different concentration for 30 minfirst and then the cells were challenged with 100 μM of H₂O₂ for 2 hoursto induce apoptosis. The apoptotic cells were calculated as thepercentage of Annexin V positive cells in total cells in FACS analysis.Recombinant human IGF-1 proteins were used as a positive control.Pre-incubation of this His-h12 recombinant protein dramatically reducedsubsequent H₂O₂-induced early apoptosis in H9C2 myocytes, resulting in a˜50% reduction in annexin V positive cells, P<0.001. The effect of HumanNo. 12 is equivalent to human recombinant IGF-1 protein at the samedose, 10 nM.

An assay was carried out to evaluate caspase inhibition by recombinantHis-h 12 protein in adult rat cardiomyocytes (FIG. 8). Adult ratcardiomyocytes were pre-incubated with 10 nM of recombinant His-h12protein for 30 min and then challenged with 100 μM of H₂O₂ for differenttime points. Cell lysates were used for the measurement of relativeamount of active caspase with Promega's Caspase-Glo kits. His-h12protein significantly reduced caspase 9 activity starting from 5 hoursonward, reaching highest inhibition (˜40% inhibition) at 9 hour,p<0.001. The absolute amount of active Caspase 3/7 is relatively lowerthan that of Caspase 9 in there cells, however, His-h12 protein alsosignificantly reduced caspase 3/7 activity at 9 hours, p<0.01.

Survival signaling mechanism of His-h12 protein on cardiomyocytes wasalso evaluated. Experiments were carried out to determine whetherHis-h12 exerts its protective effect for H₂O₂-induced apoptosis ofcardiomyocytes, in a paracrine fashion mainly through intracellularsurvival signaling transduction, which positively regulates the wholemachinery of apoptosis network. The expression of apoptosis-relatedgenes was studied in rat adult cardiomyocytes after incubation ofHis-h12 protein at 10 nM at various time points. Adult ratcardiomyocytes were incubated with recombinant His-h12 protein at 10 nMfinal concentration for 10 min, 30 min, 1 h, 2 h and 3 h. Whole celllysates were separated on 10% SDS-PAGE gels and probed with phosphor-Aktantibodies, total Akt antibody and GSK-3β antibody (FIG. 9). Lane 1,vehicle PBS control treatment; Lane 2-6, 10 min, 30 min, 1 h, 2 h and 3h incubation of cardiomyoctyes with His-h12 protein respectively.Compared with lane 1 vehicle PBS control treatment, incubation ofHis-h12 protein dramatically activates phosphor Akt^(Thr308) at 30 min,with the concurrent phosphorylation of Akt's substrate-GSK-313, at thesame time point. No significant changes were found in total Akt andβ-tublin as loading controls.

FIG. 10 shows the results of an assay to evaluate cytochrome C release.Adult rat cardiomycytes were pre-incubated with recombinant His-h12protein at 10 nM for 30 min, then challenged with 100 μM of H₂O₂ for 6 hto induce apoptosis. Cytosolic proteins were separated by 15% SDS-PAGEgel and probed with anti-cytochrome C antibodies. Lane 1-2, vehicle PBScontrol treatment; Lane 3-4, H₂O₂ treatment of cardiomyocytes for 6 h;Lane 5-8, cardiomyoctyes pre-incubated with His-h12 protein for 30 minand then challenged with H₂O₂ for 6 h. Compared with Lane 1-2 controls,H₂O₂ treatment of Lane 3-4 resulted in a dramatic release of cytochromeC into cytosolic compartment of cardiomyocytes. However, pre-incubationof His-h12 protein with cardiomyocytes for 30 min significantlyprevented the release of cytochrome C.

FIG. 11 shows stabilization of mitochondrial Bcl-2 protein level byHis-h12 protein during cardiomyocyte apoptosis. Adult rat cardiomycyteswere pre-incubated with recombinant His-h12 protein at 10 nM for 30 min,then challenged with 100 μM of H₂O₂ for 6 h to induce apoptosis.Mitochondrial proteins were separated by 12.5% SDS-PAGE gel and probewith anti-Bcl-2 antibody. Lane 1, no treatment control; Lane 2,cardiomyocytes challenged with 100 μM of H₂O₂ for 6 h; Lane 3-6,cardiomyocytes pre-incubated with 10 nM of His-h12 for 30 min and thenchallenged with 100 μM of H₂O₂ for 6 h. Compared with Lane 1 control,H₂O₂ treatment of Lane 2 resulted in a modest decrease of mitochondrialBcl-2. Pre-incubation of cardiomyocytes with His-h12 protein stabilizedthe mitochondrial Bcl-2 protein level.

Sequences and GenBank Accession Number of His-h12

No. 12 has a GenBank designation human BC037293. This gene product isalso know as chromosome 3 open reading frame 58 (c3orf58). Mouse No. 12homologous gene is currently unknown. The cDNA of human No. 12 clone waspurchased from ATCC, coding region were amplified to make the expressionconstruct, N-terminal signal deletion coding sequence of human No. 12were amplified and clone into pMal-C vector for fusion proteinexpression and purification, which were used in the initial screeningstudies. Human No. 12 was further expressed as 6×His tagged recombinantprotein for cardio-protection studies.

Human No. 12 full-length mRNA sequence (h12; SEQ ID NO 16) 1gccggagtcg gagggcgggg agctaggagg agggagctcgagagttgtgg agactagtga 61ctgggagaag tcgcagcccgctcaggcccg cgccttcccg ctccccgtct tcctctctca 121cacacctact ccgccctccgccccagcccg cgcgctagct ccttctctcg cccggggttc 181ctgccggtag ctctccgggtcttggcgcgg cgggggcgcc ccgggggtgc cctcgccctc 241ccgttgcggg cgggcgggcg gtatgtggcgcctggtgccc ccgaagctgg gccgcctgtc 301ccgctcgctg aagctggcggcgctgggcag cctgttggtg ctgatggtgc tgcactcgcc 361gtcgctgctc gcctcttggcagcgcaacga actgaccgac cggcgcttcc tgcagctcaa 421taagtgcccg gcgtgcttcg gcacgagctggtgccgccgc ttcctcaacg ggcaggtggt 481attcgaggcg tggggccgcttgcgcctgct ggacttcctc aacgtgaaga acgtgtactt 541cgcgcagtac ggcgagccccgcgagggcgg ccgccgccga gtggtgctca agcgcctcgg 601ctcgcagcgc gagctggcgcagctcgacca gagcatctgc aagcgggcca ccggccggcc 661ccgctgcgac ctgctgcaggccatgccccg gaccgagttc gcgcgcctca acggcgacgt 721gcgtctgctc acgcccgaggcggtggaggg ctggtcggac ctggtgcact gcccctcgca 781gcgccttctc gaccgcctggtgcgccgcta cgcggagacc aaggactcgg gcagcttcct 841gcttcgcaac ctcaaggactcggagcgcat gcagctgctg ctgaccctgg ccttcaaccc 901cgagccgctg gtgctacagagttttccgtc tgatgaaggt tggccatttg caaagtatct 961tggagcttgt ggaagaatggtggctgtaaa ttatgttgga gaagaactgt ggagttactt 1021taatgcgcca tgggaaaaacgagttgacct cgcttggcaa ttaatggaaa tagcagaaca 1081gcttacaaac aatgactttgaatttgcact ctacctcctg gacgtcagct ttgacaattt 1141tgcagttggt cctagagatgggaaggtaat cattgtggat gctgaaaatg ttttggttgc 1201tgacaaaaga ttaattagacaaaataaacc tgaaaattgg gatgtatggt atgaaagcaa 1261gtttgatgac tgtgataaggaggcttgctt atcattttca aaagaaattc tttgtgctcg 1321tgccactgtg gaccacaatt actatgctgt ttgtcagaac ctcttatcca gacatgccac 1381ctggcgtggc acttctggag gactccttca tgatccacca agtgaaattg ccaaagatgg 1441ccggctcgag gccttgctgg atgagtgtgc caacccaaag aagcgctatg gcagattcca 1501ggctgcaaaa gaactgcgtg aatacctagc acaattaagt aacaacgtga ggtagtctat 1561ggtgaacttt tctttttttc tccatttaaa cagcactggc taaaactaaa ccaccaaaaa 1621acgatctgaa aaaatgaaat ttggaagtgt tacattcaga ggatgataaa cttgcactga 1681tagatcttaa tgttaacatc catcaaaata agacattact tcaaaaatca catgatgctt 1741ctgcaaataa gtatgttctt atactttgga ggcttgagct gtcatcagct gctccccact 1801accccggaat gcttgagtgg attaatgaat attgttaagc tattggaaat gagtctgata 1861gtacattggc ttgtgtatca aagggtactt ggtacttagt ttgcatttac tatcatgatt 1921ttgtgaatct cttgcattta ctttgaatgt caagtcagat tggtctgttt tataggccgc 1981tttttccttc tgatgtgtag ggttttttcc cccttttttt ttttaattaa attttgaaaa 2041ttcaggttac tgtaggtgtt catttaaatt tttaatagtt gtcattcagt gctatttggt 2101acatatttac tgttagggca ggattcccag gtttactgtg tttttttttt ttttttttta 2161aagaaagcta aatattacat tatgtaaata cttcttttca ccaacttctg tagtttcacc 2221attgcatggt gtcatttcag gttatttaac agttatatcc ctctatgcca ataattagaa 2281gtgtacacta aacatgaagt ttggcatatg ttgcaaaatg tcattttatc tttctaaagg 2341ctttaagaag aatatactag aatctatata ttgatgttaa ttttgattca gaaaaaaaat 2401acaacccagt atctaaaaag tgttaactag tccaagatag taatgcatat gccaaagaaa 2461tattacacct aatctcatgt ttagaattta aaatagaatt ggtcagctac ttattcttac 2521caccctactt ccagtatttt agctctgtca ttattaaatt cagatcttcc tgattatttt 2581ttctgttgaa agttaaacta ctgctttcaa gtaatttaaa gttatcctac cttttattca 2641tgggtagttt tgcaaaatta acatggtagc cattgtttga atttaatcgg gcatcataac 2701ttttcattta ttgaggaact aatcattatt actataaagc atacaaatta gccagtcagc 2761acactttggt cttctttacc taagggttaa acatcagaac atcaaattta attatttgca 2821tagaaatgtg tgggctcttt atataagttg actatcacta acaggtaata tttttctgtt 2881tgaagttgtt acttttgttt acagcaaagt ttgatgtagt gtgcagtagt gagctctaga 2941ctgatctttt tctaaatcag aaagtgatta aagtatgcac aaccaaaggc aggtttttct 3001ttttcattta ttcagcaact atttattaag catcaactct gtgccaggca cgttactagc 3061tgctacatac tgtctgaaca tgacatacgg ttaagtaact ttacaattat tatcaaatac 3121ttcaatgtag atatttctta agttgaaata gcattaacta ggataatgct ttcatgttat 3181tttattgtct tgtgatagaa attcaacttg taccatctaa aactaggttg ctataaaaat 3241aggaggatga agtcaataaa gtttatgcca gtttaaaaac tggaaggaaa aggtaagagc 3301tctccattat aaaatagttg cattcggtta atttttacac attagtgcat tgcgtatatc 3361aactggccct caatgaagca tttaagtgct tggaatttta ctaaactgac ttttttgcaa 3421ctttgggaga tttttgaggg gagtgttgaa aattgccaaa cactcacctc ttactcaaaa 3481cttcaaataa aatacacatt ttcaagaggg agcacctttt atatttgata agttttcatt 3541ataaacctta taataccagt cacaaagagg ttgtctgtct atggtttagc aaacatttgc 3601ttttcttttt ggaagtgtga ttgcaattgc agaacagaaa gtgagaaaac actgccagcg 3661gtgattgcta cttgaggtag ttttttacaa ctaccatttc ccctccatga aattatgtga 3721aatttatttt atctttggga aaagttgaga agatagtaaa agaattagga atttaaaatt 3781acagggaaaa atatgtaagt gaaaagcaat aaatattttg ttcactttgc tatcaagatg 3841ttcactatca gatatttatt atatggcagc aatttatatt tttaatcatt gcccattaat 3901agacgcagta aaatattttt gaatcagaca tttggggttt gtatgtgcat taaaattgtc 3961ttttgtactg taagttactg ttaatttgaa tattttattg aactgtctcc ctgtgccttt 4021ataatataaa gttgtttcta caacttttaa tgatcttaat aaagaatact ttaggaaaaa 4081aaaaaaaaaa aHuman No. 12 protein sequence (sequence in underlined type was used to generaterecombinant His-h12 protein) (h12; SEQ ID NO: 17)MWRLVPPKLGRLSRSLKLAALGSLLVLMVLHSPSLLASWQRNELTDRRFLQLNKCPACFGTSWCRRFLNGQVVFEAWGRLRLLDFLNVKNVYFAQYGEPREGGRRRVVLKRLGSQRELAQLDQSICKRATGRPRCDLLQAMPRTEFARLNGDVRLLTPEAVEGWSDLVHCPSQRLLDRLVRRYAETKDSGSFLLRNLKDSERMQLLLTLAFNPEPLVLQSFPSDEGWPFAKYLGACGRMVAVNYVGEELWSYFNAPWEKRVDLAWQLMEIAEQLTNNDFEFALYLLDVSFDNFAVGPRDGKVIIVDAENVLVADKRLIRQNKPENWDVWYESKFDDCDKEACLSFSKEILCARATVDHNYYAVCQNLLSRHATWRGTSGGLLHDPPSEIAKDGRLEALLDECANPKKRYGRFQAAKELREYLAQLSNNVROther genes and gene products, the function and activity of which havepreviously not been known, have now been identified as havingcardioprotective activity. The nucleic acid and amino acid sequences ofthese factors are described below.

Human No. 1 mRNA sequence (h1: SEQ ID NO: 18) 1gcatcttggc agggtccggg gacgtggactatttcgcacaccacaccacg gggagggatt 61tttttctatt ttccctacgaaaaacagatc tttttaaggatggtgctgct ccactggtgc 121ctgctgtggc tcctgtttccactcagctca aggacccagaagttacccac ccgggatgag 181gaactttttc agatgcagatccgggacaag gcattttttcatgattcgtc agtaattcca 241gatggagctg aaattagcagttatctcttt agagatacacctaaaaggta tttctttgtg 301gttgaagaag acaatactccattatcagtc acagtgacgccctgtgatgc gcctttggag 361tggaagctga gcctccaggagctgccagag gacaggagcggggaaggctc aggtgatctg 421gaacctcttg agcagcagaagcagcagatc attaatgaggaaggcactga gttattctcc 481tacaaaggca atgatgttgagtattttata tcgtctagttccccatccgg tttgtatcag 541ttggatcttc tttcaacagagaaagacaca catttcaaagtatatgccac cacaactcca 601gaatctgatc agccataccctgagttaccc tatgacccaagagtagatgt gacctcactg 661gggcgcacca cggtcactttggcctggaaa ccaagccccactgcctcttt gctgaaacaa 721cccattcagt actgtgtggtcatcaacaaa gagcacaatttcaaaagtct ctgtgcagtg 781gaagcaaaac tgagtgcagatgatgctttt atgatggcaccgaaacctgg tctggacttc 841agcccctttg actttgcccactttggattt ccttctgataattcaggtaa agaacgcagt 901ttccaggcaa agccttctccaaaactgggg cgtcatgtctactccaggcc caaggttgat 961attcagaaaa tctgcataggaaacaagaac atcttcaccgtctctgatct gaaacccgac 1021acgcagtact actttgatgtatttgtggtc aacatcaacagcaacatgag caccgcttat 1081gtaggtacct ttgccaggac caaggaagaa gccaaacaga agacagtc gagctaaaagat 1141gggaagataa cagatgtatt tgttaaaagg aagggagcaa agtttcta cggtttgctcca 1201gtctcttctc accaaaaagt caccttcttt attcactctt gtctggat gctgtccaaatc 1261caagtgagaa gagatgggaa acttcttctg tctcagaatg tggaaggc attcagcagttt 1321cagcttagag gaaaacctaa agctaaatac ctcgttcgac tgaaagga aacaagaaagga 1381gcatctatgt tgaaaattct agctaccaca aggcctacta agcagtca tttccctctctt 1441cctgaagaca caagaatcaa agcctttgac aagctccgta cctgttcc tcggccaccgtg 1501gcttggctag gcactcagga aaggaacaag ttttgcatct acaaaaaa gaagtggatgat 1561aactacaatg aagaccagaa gaaaagagag caaaaccaat gtctagga ccagatataagg 1621aagaagtcag aaaaggtcct ctgtaaatat ttccacagtc aaaacttg cagaaagcagtg 1681accacagaaa caattaaagg tcttcagcct ggcaaatctt acctgctg gatgtttatgtc 1741ataggacatg gggggcactc tgtaaagtat cagagtaagg ttgtgaaa actagaaagttc 1801tgttagttac cttcttatag agatatatta tgtagaactc caggaggg acattaaatcac 1861tttaagtata aactgactac tcccacagtt gagagaagtt gtgacctg tacttgtactat 1921ggaaggaagg atatcaacgt gtgtatattg atgtttatat aagtaact cttgaaggagac 1981ttgttctagc gtgccccatg gtacctagtg tgtgtctgat gccggttg gtgtcaaagata 2041gagggcttct tgaaggaact tgccattcct tgctttgacc actgcatg aactgcttctaa 2101attattttat tacctaaaaa tttaaaatat gccattcatt gcacacac ccacaaatgcaa 2161atcattcctc tctatagatg ctaggatata tataaattat tttataaa ttcttgttttaa 2221atgtcagtgt ttctatgatt gtaaactatt aaattctttt cctattaa agtacagatcta 2281atctaagtat tattaagttg atagccctct agtcagttat attgctat tgtaaattcttg 2341tttgttgagt aaaatgttta aatactatat gtatctcatg tacaaagt tgacatacatta 2401tattcatgta cataaaatta aagagattag attataaHuman No. 1 protein sequence (h1: SEQ ID NO: 19)MVLLHWCLLWLLFPLSSRTQKLPTRDEELFQMQIRDKAFFHDSSVIPDGAEISSYLFRDTPKRYFFVVEEDNTPLSVTVTPCDAPLEWKLSLQELPEDRSGEGSGDLEPLEQQKQQIINEEGTELFSYKGNDVEYFISSSSPSGLYQLDLLSTEKDTHFKVYATTTPESDQPYPELPYDPRVDVTSLGRTTVTLAWKPSPTASLLKQPIQYCVVINKEHNFKSLCAVEAKLSADDAFMMAPKPGLDFSPFDFAHFGFPSDNSGKERSFQAKPSPKLGRHVYSRPKVDIQKICIGNKNIFTVSDLKPDTQYYFDVFVVNINSNMSTAYVGTFARTKEEAKQKTVELKDGKITDVFVKRKGAKFLRFAPVSSHQKVTFFIHSCLDAVQIQVRRDGKLLLSQNVEGIQQFQLRGKPKAKYLVRLKGNKKGASMLKILATTRPTKQSFPSLPEDTRIKAFDKLRTCSSATVAWLGTQERNKFCIYKKEVDDNYNEDQKKREQNQCLGPDIRKKSEKVLCKYFHSQNLQKAVTTETIKGLQPGKSYLLDVYVIGHGGHSVKYQSKVVKTRK FCHuman No. 5 mRNA sequence (h5; SEQ ID NO: 20 1agcgggatag cccgcggccg cgcctgcccg ctcgcacccc tctcccgcgc ccggttctcc 61ctcgcagcac ctcgaagtgc gcccctcgcc ctcctgctcg cgccccgccg ccatggctgc 121ctcccccgcg cggcctgctg tcctggccct gaccgggctg gcgctgctcc tgctcctgtg 181ctggggccca ggtggcataa gtggaaataa actcaagctg atgcttcaaa aacgagaagc 241acctgttcca actaagacta aagtggccgt tgatgagaat aaagccaaag aattccttgg 301cagcctgaag cgccagaagc ggcagctgtg ggaccggact cggcccgagg tgcagcagtg 361gtaccagcag tttctctaca tgggctttga cgaagcgaaa tttgaagatg acatcaccta 421ttggcttaac agagatcgaa atggacatga atactatggc gattactacc aacgtcacta 481tgatgaagac tctgcaattg gtccccggag cccctacggc tttaggcatg gagccagcgt 541caactacgat gactactaac catgacttgc cacacgctgt acaagaagca aatagcgatt 601ctcttcatgt atctcctaat gccttacact acttggtttc tgatttgctc tatttcagca 661gatcttttct acctactttg tgtgatcaaa aaagaagagt taaaacaaca catgtaaatg 721ccttttgata tttcatggga atgcctctca tttaaaaata gaaataaagc attttgttaa 781aaagaaaaaa aaaaaaaaaa Human No. 5 protein sequence (h5; SEQ ID NO: 21)MAASPARPAVLALTGLALLLLLCWGPGGISGNKLKLMLQKREAPVPTKTKVAVDENKAKEFLGSLKRQKRQLWDRTRPEVQQWYQQFLYMGFDEAKFEDDITYWLNRDRNGHEYYGDYYQRHYDEDSAIGPRSPYGFRHGASVNYDDYHuman No. 8 mRNA sequence (h8; SEQ ID NO: 22) 1cactgggaga cagtccactt aaatgcagct ccagggttgc gaggcaccca ccagcatcat 61tccccatgcg aggtggcaaa tgcaacatgc tctccagttt ggggtgtcta cttctctgtg 121gaagtattac actagccctg ggaaatgcac agaaattgcc aaaaggtaaa aggccaaacc 181tcaaagtcca catcaatacc acaagtgact ccatcctctt gaagttcttg cgtccaagtc 241caaatgtaaa gcttgaaggt cttctcctgg gatatggcag caatgtatca ccaaaccagt 301acttccctct tcccgctgaa gggaaattca cagaagctat agttgatgca gagccgaaat 361atctgatagt tgtgcgacct gctccacctc caagtcaaaa gaagtcatgt tcaggtaaaa 421ctcgttctcg caaacctctg cagctggtgg ttggcactct gacaccgagc tcagtcttcc 481tgtcctgggg tttcctcatc aacccacacc atgactggac attgccaagt cactgtccca 541atgacagatt ttatacaatt cgctatcgag aaaaggataa agaaaagaag tggatttttc 601aaatctgtcc agccactgaa acaattgtgg aaaacctaaa gcccaacaca gtttatgaat 661ttggagtgaa agacaatgtg gaaggtggaa tttggagtaa gattttcaat cacaagactg 721ttgttggaag taaaaaagta aatgggaaaa tccaaagtac ctatgaccaa gaccacacag 781tgccagcata tgtcccaagg aaactaatcc caataacaat catcaagcaa gtgattcaga 841atgttactca caaggattca gctaaatccc cagaaaaagc tccactggga ggagtgatac 901tagtccacct tattattcca ggtcttaatg aaactactgt aaaacttcct gcatccctaa 961tgtttgagat ttcagatgca ctcaagacac aattagctaa gaatgaaacc ttggcattac 1021ctgccgaatc taaaacacca gaggttgaaa aaatctcagc acgacccaca acagtgactc 1081ctgaaacagt tccaagaagc actaaaccca ctacgtctag tgcattagat gtttcagaaa 1141caacactggc ttcaagtgaa aagccatgga ttgtgcctac agctaaaata tctgaagatt 1201ccaaagttct gcagcctcaa actgcaactt atgatgtttt ctcaagccct acaacatcag 1261atgagcctga gatatcagat tcctacacag caacaagtga tcgtattctg gattctatcc 1321cacctaaaac ttctagaact cttgaacagc caagggcaac actggctcca agtgaaacac 1381catttgttcc tcaaaaactg gaaatcttta ccagtccaga aatgcagcct acgacacctg 1441ctccccagca aactacatct atcccttcta cacctaaacg acgcccccgg cccaaaccgc 1501caagaaccaa acctgaaaga accacaagtg ccggaacaat tacacctaaa atttctaaaa 1561gccctgaacc tacatggaca acaccggctc ccggtaaaac acaatttatt tctctgaaac 1621ctaaaatccc tctcagccca gaagtgacac acaccaaacc tgctcccaag cagacaccac 1681gtgctcctcc taagccaaaa acatcaccac gcccaagaat cccacaaaca caaccagttc 1741ctaaggtgcc ccagcgtgtt actgcaaaac caaaaacgtc accaagtcca gaagtgtcat 1801acaccacacc tgctccaaaa gatgtgctcc ttcctcataa accataccct gaggtctctc 1861agagcgaacc tgctcctcta gagacacgag gcatcccttt tatacccatg atttccccaa 1921gtcctagtca agaggaacta cagaccactc tggaagaaac agaccaatcc acccaagaac 1981ctttcacaac taagattcca cgaacaactg aactagcaaa gacaactcag gcgccacaca 2041gattttatac tactgtgagg cccagaacat ctgacaagcc acacatcaga cctggggtca 2101agcaagcacc caggccatca ggtgctgata gaaatgtatc agtggactct acccacccca 2161ctaaaaagcc agggactcgc cgcccaccct tgccacccag acctacacac ccacgaagaa 2221aacctttacc accaaataat gtcactggaa agccaggaag tgcaggaatc atttcatcag 2281gcccaataac tacaccaccc ctgaggtcaa cacccaggcc tactggaact cccttggaga 2341gaatagagac agatataaag caaccaacag ttcctgcctc tggagaagaa ctggaaaata 2401taactgactt tagctcaagc ccaacaagag aaactgatcc tcttgggaag ccaagattca 2461aaggacctca tgtgcgatac atccaaaagc ctgacaacag tccctgctcc attactgact 2521ctgtcaaacg gttccccaaa gaggaggcca cagaggggaa tgccaccagc ccaccacaga 2581acccacccac caacctcact gtggtcaccg tggaagggtg cccctcattt gtcatcttgg 2641actgggaaaa gccactaaat gacactgtca ctgaatatga agttatatcc agagaaaatg 2701ggtcattcag tgggaagaac aagtccattc aaatgacaaa tcagacattt tccacagtag 2761aaaatctgaa accaaacacg agttatgaat tccaggtgaa acccaaaaac ccgcttggtg 2821aaggcccggt cagcaacaca gtggcattca gtactgaatc agcggaccca agagtgagtg 2881agccagtttc tgcaggaaga gatgccatct ggactgaaag accctttaat tcagactctt 2941actcagagtg taagggcaaa caatatgtca aaaggacatg gtataaaaaa tttgtaggag 3001tgcagctgtg caactctctc agatacaaga tttacttgag cgactccctc acaggaaaat 3061tttataacat aggtgatcag aggggccatg gagaagatca ctgccagttt gtggattcat 3121ttttagatgg acgcactggg cagcaactca cttctgacca gttaccaatc aaagaaggtt 3181atttcagagc agttcgccag gaacctgtcc aatttggaga aataggtggt cacacccaaa 3241tcaattatgt tcagtggtat gaatgtggga ctacaattcc tggaaaatgg tagatgctgc 3301acaaagttac cttctgtttc atcattgcaa acaaaaatca ttgaaaatac tatgccgcat 3361tcatttaaag ctattttgtt tactatgtat aaaagtctac aatctaatta atagcaatac 3421tagatgttta ttattagaaa agattgctga gagtatttat caggttttac aaagtcattt 3481taagaaagca agatactgat gttaacagaa taacattttt ggggaagctg gctccctatt 3541catggtattt taagagatca tttgtatatt atttatcaca ctgttgtaat gatgttttga 3601gatactttta taacaaaatt aacatcaaaa aggtatatac tttttaaaaa aaatttactt 3661ttattgatgt gtactcttcc tattgatgag ttaattccat aaatctctac ttagtttaac 3721ttattggatc aaattatctt cagcatgtat atctggggaa aaaaggtccg aattttcaca 3781tttatattta aacttcaatt ttttatattt aaacttcaat tttttagcaa cagctgaata 3841gctttgcgga ggagtttaat agttacacat tcatgctaat atacatttcc tttaaacatc 3901cacaaattct taaaaagatt gaatcagtaa atttcatttc agctaaaaat ggagtctaat 3961atattgtttc aaaagataca tttttaccca ccataaatgt tacaatatct gaatatgctt 4021tgtcaaacta tccctttatg caatcgtctt catattgttt ttatgattct aatcaagctg 4081tatgtagaga ctgaatgtga agtcaagtct gagcacaaaa agataatgca caatgagatt 4141gcctaccatt ttataggata tttactatgt atttatacgt taagacctct atgaatgaat 4201gtatcagaga atgtctttgt aactaactgt ttaattcaat ctgtaataaa aatctaacta 4261actaactcat ttatttctat taaaaaggta ttgtccttta ggcggggaat gggaatcctt 4321gctgcactgt tgcagtcatt ctgaaaggac ctttccctgt acttaccttt caacatgctt 4381caatcttatc aacgctacat tttgtatttt tcaaacaggt ataaattctg caataaagag 4441atgtagtttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4501aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaHuman No. 8 protein sequence (h8; SEQ ID NO: 23)MRGGKCNMLSSLGCLLLCGSITLALGNAQKLPKGKRPNLKVHINTTSDSILLKFLRPSPNVKLEGLLLGYGSNVSPNQYFPLPAEGKFTEAIVDAEPKYLIVVRPAPPPSQKKSCSGKTRSRKPLQLVVGTLTPSSVFLSWGFLINPHHDWTLPSHCPNDRFYTIRYREKDKEKKWIFQICPATETIVENLKPNTVYEFGVKDNVEGGIWSKIFNHKTVVGSKKVNGKIQSTYDQDHTVPAYVPRKLIPITIIKQVIQNVTHKDSAKSPEKAPLGGVILVHLIIPGLNETTVKLPASLMFEISDALKTQLAKNETLALPAESKTPEVEKISARPTTVTPETVPRSTKPTTSSALDVSETTLASSEKPWIVPTAKISEDSKVLQPQTATYDVFSSPTTSDEPEISDSYTATSDRILDSIPPKTSRTLEQPRATLAPSETPFVPQKLEIFTSPEMQPTTPAPQQTTSIPSTPKRRPRPKPPRTKPERTTSAGTITPKISKSPEPTWTTPAPGKTQFISLKPKIPLSPEVTHTKPAPKQTPRAPPKPKTSPRPRIPQTQPVPKVPQRVTAKPKTSPSPEVSYTTPAPKDVLLPHKPYPEVSQSEPAPLETRGIPFIPMISPSPSQEELQTTLEETDQSTQEPFTTKIPRTTELAKTTQAPHRFYTTVRPRTSDKPHIRPGVKQAPRPSGADRNVSVDSTHPTKKPGTRRPPLPPRPTHPRRKPLPPNNVTGKPGSAGIISSGPITTPPLRSTPRPTGTPLERIETDIKQPTVPASGEELENITDFSSSPTRETDPLGKPRFKGPHVRYIQKPDNSPCSITDSVKRFPKEEATEGNATSPPQNPPTNLTVVTVEGCPSFVILDWEKPLNDTVTEYEVISRENGSFSGKNKSIQMTNQTFSTVENLKPNTSYEFQVKPKNPLGEGPVSNTVAFSTESADPRVSEPVSAGRDAIWTERPFNSDSYSECKGKQYVKRTWYKKFVGVQLCNSLRYKIYLSDSLTGKFYNIGDQRGHGEDHCQFVDSFLDGRTGQQLTSDQLPIKEGYFRAVRQEPVQFGEIGGHTQINYVQWYECGTTIPGKWHuman No. 13 mRNA sequence (h13; SEQ ID NO: 24) 1ctccggtgag ttttgtggcg ggaagcttct gcgctggtgc ttagtaaccg actttcctcc 61ggactcctgc acgacctgct cctacagccg gcgatccact cccggctgtt cccccggagg 121gtccagaggc ctttcagaag gagaaggcag ctctgtttct ctgcagagga gtagggtcct 181ttcagccatg aagcatgtgt tgaacctcta cctgttaggt gtggtactga ccctactctc 241catcttcgtt agagtgatgg agtccctaga gggcttacta gagagcccat cgcctgggac 301ctcctggacc accagaagcc aactagccaa cacagagccc accaagggcc ttccagacca 361tccatccaga agcatgtgat aagacctcct tccatactgg ccatattttg gaacactgac 421ctagacatgt ccagatggga gtcccattcc tagcagacaa gctgagcacc gttgtaacca 481gagaactatt actaggcctt gaagaacctg tctaactgga tgctcattgc ctgggcaagg 541cctgtttagg ccggttgcgg tggctcatgc ctgtaatcct agcactttgg gaggctgagg 601tgggtggatc acctgaggtc aggagttcga gaccagcctc gccaacatgg cgaaacccca 661tctctactaa aaatacaaaa gttagctggg tgtggtggca gaggcctgta atcccagctc 721cttgggaggc tgaggcggga gaattgcttg aacccgggga cggaggttgc agtgagccga 781gatcgcactg ctgtacccag cctgggccac agtgcaagac tccatctcaa aaaaaaaaaa 841aaaaaaaaaa aaaaaaaaa Human No. 13 protein sequence (h13; SEQ ID NO: 25)MKHVLNLYLLGVVLTLLSIFVRVMESLEGLLESPSPGTSWTTRSQLANTEPTKGLPDHPSRSM

Other Embodiments

Although particular embodiments have been disclosed herein in detail,this has been done by way of example for purposes of illustration only,and is not intended to be limiting with respect to the scope of theappended claims, which follow. In particular, it is contemplated by theinventors that various substitutions, alterations, and modifications maybe made to the invention without departing from the spirit and scope ofthe invention as defined by the claims. The choice of nucleic acidstarting material, clone of interest, or library type is believed to bea matter of routine for a person of ordinary skill in the art withknowledge of the embodiments described herein.

Other aspects, advantages, and modifications considered to be within thescope of the following claims.

1. A method of reducing cell death, comprising contacting an injured ordiseased tissue with a composition comprising a purified paracrinefactor of a mesenchymal stem cell (MSC), wherein said factor comprisesSEQ ID NO:17 or a fragment thereof and wherein said tissue comprisesneurological tissue.
 2. The method of claim 1, wherein said compositioncomprises a mixture of at least two paracrine factors.
 3. The method ofclaim 1, wherein said tissue is characterized by ischemic or reperfusioninjury.
 4. The method of claim 1, wherein said composition furthercomprises a compound selected from the group consisting of Sfrp-1,Sfrp-2, and Sfrp-3.
 5. The method of claim 4, wherein said Sfrp-1comprises an amino acid sequence of SEQ ID NO:5, a mature processed formof SEQ ID NO:5, or a fragment thereof.
 6. The method of claim 4, whereinsaid Sfrp-2 comprises an amino acid sequence of SEQ ID NO:7, a matureprocessed form of SEQ ID NO:7, or a fragment thereof.
 7. The method ofclaim 4, wherein said Sfrp-3 comprises an amino acid sequence of SEQ IDNO:9, a mature processed form of SEQ ID NO:9, or a fragment thereof. 8.(canceled)
 9. The method of claim 1, wherein the amount of apoptoticcell death is reduced in the presence of said factor compared to in itsabsence. 10-11. (canceled)
 12. The method of claim 1, wherein saidinjured or diseased tissue is associated with a disorder selected fromthe group consisting of ischemic disorders, reperfusion relateddisorders, and stroke.
 13. The method of claim 1, wherein saidcomposition comprises a slow-release formulation.
 14. The method ofclaim 1, wherein said composition is systemically administered.
 15. Themethod of claim 1, wherein said composition is locally administered tosaid tissue.
 16. The method of claim 1, wherein said composition isadministered to said tissue prior to an ischemic event orischemia-reperfusion injury.
 17. The method of claim 1, wherein saidcomposition is administered at the at the time of ischemia or ischemiaor reperfusion injury.
 18. The method of claim 1, wherein saidcomposition is administered after an ischemic event or ischemia orreperfusion injury.
 19. The method of claim 1, wherein said compositionfurther comprises a factor comprising the amino acid sequence of SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25.
 20. The method ofclaim 1, wherein said composition is administered in an amount thatreduces apoptotic cell death.
 21. The method of claim 1, wherein saidfactor comprises amino acids 45-430 of SEQ ID NO:17.
 22. The method ofclaim 1, wherein said neurological tissue comprises brain or spinalcord.